international symposium on earthworm ecology 5 - archive

1

The 9th International Symposium on Earthworm Ecology

5th to 10

th September 2010, Xalapa, Mexico

The 9th

International

Symposium on Earthworm Ecology

5th

– 10th

September 2010

Xalapa, Mexico

2


5th to 10


CONTENTS

Introductory and Magistral lecture 3

Session 1 – Earthworm Biodiversity & DNA Bar-Coding 4

Oral Contributions 5

Poster Contributions 16

Session 2 – Earthworm Immunology & Physiology 42



Session 3 – Earthworms as Indicators of Change on a Local, Regional & Global Scale 83



Session 4 – Earthworms & Ecosystem Services 121

Oral Contributions 122 Poster Contributions 132

Session 5 – Earthworms Interaction 158

Oral Contributions 159 Poster Contributions 170

Session 6 – Earthworms, Soil & Fertility 204



Session 7 – Earthworms, Soil Contamination & Bioremediation 232



Session 8 – Vermicomposting & Waste Management 265



Author Index 305

3


5th to 10


Introductory and Magistral Lecture

Earthworms, Gaia's best friends

Lavelle Patrick

Université P. et M. Curie (Paris 6), UMR BIOEMCO 7618

Centre IRD Ile de France, CIAT/TSBF

Cali, Colombia

It is said that worms went out from the sea 500M years ago and colonised terrestrial environments.

Since then, earthworms known for at least 100M years, have developped a large diversity of life

forms distributed among several thousands of species able to exploit any ecological niche in the soil

and litter environment. They have developped intense interactions with all soil organisms through

their remarkable activity as soil ecosystem engineers. They have also become efficient protectors of

plants through still little understood mechanisms. We only know so far that very intimate interactions

involving plant gene expression are at hand. As a result, earthworms are major providers of soil

ecosystem engineers.

Earthworms must have gone through a number of major disturbances and changes. The shift from

Gymnosperm to Angiosperms dominant vegetation must have profoundly affected the quality of

organic inputs in litter and soils. Closer to us, they lived the great Quaternary glaciations and

recolonised the newly formed soils after retreat. The Anthropocene is now at hand. Many earthworm

species will likely disappear as a result of conversion or modification of their natural habitat.

However, a small number of peregrine species, specially adapted to face great disturbances are

already working to limit damages to soils and plant communities, and the remarkable ability of the

group to create new species will likely replace the extinct pool of inadapted species. Earthworms will

remain Gaias's best friends and help go through one more disturbance.

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5th to 10


Session 1

Earthworm Biodiversity

and DNA Bar-coding

5


5th to 10


Plenary Lecture – Session 1

Molecular data on the point of revolutionizing earthworm systematics

James Samuel

University of Kansas

From the simple questions of correct application of names to formally described taxa to testing

hypotheses of ancestral relationships and origins of faunas, molecular data are bringing clarity and

focus to the scientific investigation of earthworm diversity. These data are also upsetting some

enduring species concepts, challenging taxonomic opinions regarding synonymy, and may bring

precision at the cost of abandoning exclusive reliance on morphology for identification of

earthworms. The global earthworm DNA barcoding campaign based at the Canadian Centre for DNA

Barcoding has uncovered cryptic diversity within many common earthworm lineages, as have other

working groups. These discoveries lead to the need to match lineages to type material, and we can

anticipate that some names will be restored from synonymy. This is not always possible and the

taxonomic community may need to take action necessary to secure the future of earthworm

identification. Bayesian phylogenetic analyses of 18s + 28s sequence data reveal taxon trees that do

not correspond to classical earthworm systematics or the implicit homology statements of those

systems. These force us to evaluate morphology in different ways and to redefine higher levels of the

classification. Earthworms appear to be very old, but we need to do more to calibrate molecular

clock(s) in order to estimate dates of divergence and the present and paleo-biogeography of

earthworm higher taxa.

6


5th to 10


Oral – Session 1

Shifting clone diversity and morphometry across northern Europe in the

parthenogenetic earthworm Eiseniella tetraedra (Sav.) including samples

from sites exposed to radioactive pollution

Terhivuo Juhani

1, Halmepuro Anne-Mari

1, Saura Anssi

2

1Finnish Museum of Natural History, Zoological Museum, University of Helsinki, Finland

2Department of Molecular Biology, Umeå University, Sweden

We have earlier surveyed clone pool structure and variability of morphometrical characters in

parthenogenetic earthworms around the Baltic Sea. Here we give results on clone pool diversity and

morphometrical variation in the riparian parthenogenetic lumbricid Eiseniella tetraedra (Sav.) as

indicated by sampled specimens from Norway, Sweden, Finland and the Komi Republic bordering

the Ural mountains. A pattern of geographic displacement of clones from west to east was observed

and the overall clone diversity decreased towards the east. We also found that fresh weight and

posterior body length were lower in adults of the east than in those of the west whereas the number of

posterior segments did not differ statistically significantly between the countries. We hypothesize

that the decreasing eastward trends may indicate adaptation of populations to the more severe

continental climate of the east. There were, however, significant differences between populations

within a country perhaps attributable to local factors. As for generative characters no clear-cut

geographical west-east trend was observed. In the Vodnyi village (Komi) E. tetraedra populations

occupied top soils contaminated by exposure to long-term ionizing radiation resulting from radium

extraction from groundwater. The upstream samples of the Ukhta river running close to the

contaminated site at Vodnyi (Komi) were made up of several clones, while the samples downstream

were almost invariable, including the ones from the sites with the highest levels of background

radiation. Further studies are needed to ascertain whether this invariability is related to environmental

pollution because clone assemblages in the samples from varying distances of Vodnyi were either

variable or monomorphic.

7


5th to 10


Oral– Session 1

Phylogeographic and adaptive study in the earthworm Lumbricus rubellus

sampled from locations corresponding to southern European glacial

refugia and extending into northern Europe

Sechi Pierfrancesco

Cardiff University

Earthworms play key direct and indirect roles in many soil processes, in food web ecology, and

ecosystem functioning. For these reasons they are highly significant as bio-monitoring species. Due

to their keystone and ecological engineering status, it is essential to achieve a better knowledge and

understanding of their population structures, evolutionary histories, and genetic diversity. It is highly

likely that during the last glaciation period, the lumbricid fauna of Great Britain was completely

eradicated, with the post-glacial fauna having migrated from one or more refugia in mainland Europe

extending from Iberia to the Carpathians. A probable consequence of this can be found in the paucity

of species in Great Britain, consisting of only ~26 species. In comparison, the number of extant

species in southern mainland Europe is ~180 species. Despite the importance of representative

lumbricids in ecotoxicology, a large-scale genetic analysis of the patterns of dispersal of certain

relatively ubiquitous species from recognized glacial refugia and the subsequent (re)colonization of

northern latitudes is conspicuously lacking. The present project ultimately aims to study the

phylogeography of the acid-tolerant, cosmopolitan, epigeic species Lumbricus rubellus with regard to

the post-glacial colonization of North Europe using mitochondrial DNA markers and next-generation

sequencing tools. The project will also investigate the evolutionary processes and metabolic

responses of populations that have established sustainable footholds on polluted sites. The Cardiff

laboratory has already identified two deeply-differentiated mitochondrial lineages within L. rubellus.

The specific purpose of the pan-European collaborative initiative presented here was to explore the

mainland European origin(s) of northern including (especially) British L. rubellus populations, and to

verify the spatial/temporal clustering of the two presently recognised lineages. We present the

preliminary phylogenetic study based on mitochondrial genotype (COI and COII loci) data obtained

from specimens from sites in Great Britain, Spain, Portugal, France, Italy, Serbia, Poland, Estonia,

Denmark, Austria, Hungary, Sweden, Finland and Norway.

8


5th to 10


Oral – Session 1

The never ending story of the Aporrectodea caliginosa species complex:

new insights into its phylogeny

Fernández Rosa, Novo Marta, Gutiérrez Mónica, Almodóvar Ana, Díaz Cosín Dario J.

Universidad Complutense de Madrid

The taxonomical status of the species included in the Aporrectodea caliginosa complex has been a

matter of discussion for centuries. Recent phylogenetic analysis added useful molecular information,

showing that A. trapezoides is not a subspecies of A. caliginosa as traditionally thought, and that it is

genetically more related to A. nocturna and A. longa. In this study, we sequenced more earthworms

from the above-mentioned species, and in addition four morphologically very similar species, never

considered before to belong to the species complex, were included: A. giardi, Nicodrilus

tetramammalis, N. monticola and N. carochensis, the three last ones recently described and

considered by taxonomists as synonyms of A. caliginosa. A total of 250 earthworms collected in 47

populations from Spain, France, Portugal, Italy, Greece, Turkey, Egypt and Australia were

sequenced. More emphasis was put on the sequencing of A. trapezoides as one of the aims of this

study was to reveal the phylogeography of this parthenogenetic species (see Fernández et al., this

volume). The second aim was to study in depth the phylogeny of the complex including the above-

mentioned species. The third one was to check with molecular tools if the three above-mentioned

species of Nicodrilus were similar to A. caliginosa. Two mitochondrial (cytochrome oxidase I -456

bp- and II – 564 bp) and two nuclear (28s - 675 bp -and histone 3 -311 bp) genes were chosen as

molecular markers. Phylogenetic analyses (Neighbor-joining, Maximum parsimony,

Maximum likelihood and Bayesian) revealed two clearly separated and statistically well-

supported lineages in A. trapezoides: lineage A, in the North of Spain and France, and lineage B, in

Central and Southern Spain and the rest of the studied countries. Regarding the phylogeny of the

whole complex, lineage A of A. trapezoides clustered together with A. nocturna, A. longa and A.

giardi, suggesting that from the molecular point of view A. giardi should be included in the complex

of species. The lineage B of A. trapezoides clustered together with the newly described species of the

genus Nicodrilus. After these results the taxonomical status of A. trapezoides should be reconsidered,

as both lineages were genetically more related to different amphimictic species, suggesting a

different origin of the two lineages. In addition, there is strong molecular evidence to support that not

only the Nicodrilus species cannot be considered as synonymous of A. caliginosa, but also that they

should be included in the genus Aporrectodea, at least until a further revision supports the idea of

Nicodrilus.

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5th to 10


Oral – Session 1

The genome of the earthworm Lumbricus rubellus

Elsworth Ben1, Thompson Marian

1, Grabara Karolina

1, Montazam Anna

1, Cleven Denis

1,

Kille Peter2, Morgan John

2, Stürzenbaum Stephen R.

3, Spurgeon David

4, Svendsen Claus

4

1The University of Edinburgh

2Cardiff University

3King's College London

4Centre for Ecology & Hydrology

Our understanding of an organism's biology is greatly advanced through the study of its genome.

Previously, sequencing a metazoan genome was a major undertaking applied to a handful of model

organisms and their close relatives. However, second generation sequencing has made it possible to

produce a good-quality genome sequence for any chosen species. There are as yet no genome

sequences published for Annelida. Embracing the new sequencing technology, we have generated

and assembled a draft genome for the earthworm Lumbricus rubellus. Three wild-caught specimens

showing low genomic divergence were chosen for sequencing using Illumina SOLEXA and Roche

454 technologies. From ~40 fold coverage we have assembled an estimate of the 430 megabase

genome. We annotated the genome sequence using the extensive expressed sequence tag (EST)

resource as well as numerous computational analyses. The genome sequence is likely to be near-

complete, as we have been able to identify genomic coordinates for 90% of the ESTs. Gene

prediction softwares identify about 23,000 protein coding genes. We have used expert manual

annotation to explore key metabolic pathways, identify gene families of interest, and to map the age

and pattern of expansion of transposable elements. The genome sequence will be a springboard for

further analyses, such as genomic biology of metal and other pollutant responses, the dynamics of

epigenetic modification of the genome by methylation and definition of novel metabolic pathways.

10


5th to 10


Oral - Session 1

SeqAWorm: Sequencing a Soil Sentinel

Kille Peter

1, Elsworth Ben

2, Spurgeon David


4, Svendsen Claus

3,

Morgan John1, Blaxter Mark.

2

1Cardiff University

2University of Edinburgh

3Centre of Ecology and Hydrology, UK

4King's College, London

Next generation sequencing has been harnessed to generate a draft of the 440Mb of the Lumbricus

rubellus genome, heralding a new era in our understanding of oligochaete biology. This resource is

providing immediate insight into the functional components that drive earthworm biology and

ecology. The presence of conserved essential metabolic pathways is contrasted by the identification

of homologues to proteins associated with vertebrate hearing and sight which may underlie

earthworm sensitivity to vibration and light. Further comparative genetics has identified the gene

encoding earthworm chitin which plays an essential role in the structure of chaetae and in providing a

non-abrasive surface in the crop and gizzard. We have overlain the genome with a range of cDNA

libraries generated from other representative members of the Lumbricidae using classical expressed

sequence tag (EST) approaches, subtractive techniques, and next generation technologies. This

exercise has yielded novel insights that are significantly enriching the functional aspect of the

genome sequence resource, whilst also serving as a framework for intra- and inter- library

comparisons and gene-object association. The future assembly of the draft genome into a physical

map associated with karyotype, together with the generation of high density SNP maps, will allow

the rapid and effective identification of the genetic basis of observed phenotypes. Furthermore, the

genome also provides the basis from which to probe the relationship between the epigenome and the

environment in organisms with a body plan and mode of existence that contribute to an intimate

relationship between the animal and its substrate. However, far from being the end of a process this

collaborative earthworm sequencing venture provides the scaffold on which to build a deeper

understanding of the molecular mechanisms that drive fundamental aspects of the endocrinology,

ecology, evolution and ecotoxicology of a taxon whose ecological engineering and sentinel status is

widely acknowledged. Acknowledgement: This initiative was funded by a research grant

(NE/F001185/1) awarded by the UK Natural Environment Research Council.

11


5th to 10


Oral - Session 1

The taxonomic puzzle of Hormogastridae earthworms: molecular insights

Novo Marta1, Fernández Rosa

1, Gutiérrez Mónica

1, Almodóvar Ana

1, Giribet Gonzalo

2,

Díaz Cosín Dario J.1

1Universidad Complutense de Madrid

2Harvard University

The classical taxonomy of earthworms is somehow limited due to their anatomical simplicity and the

variability of their characteristics often used for diagnosing species. In this study we use molecular

tools to unravel the phylogeny of Hormogastridae. This family includes four genera and 22 species

(and eight subspecies or varieties) of large to middle-sized earthworms distributed almost exclusively

in the countries and islands of the western Mediterranean where they play a very important

ecological role. The genus Hormogaster includes 18 of the 22 described species and the Iberian

Peninsula is host to the highest diversity, with 16 species located in central and eastern Spain. We

collected 375 individuals representing the four described genera and 20 species in 46 different

locations from the Iberian Peninsula, France, Corsica and Sardinia. Molecular markers included

mitochondrial regions of the cytochrome c oxidase subunit I gene (COI), 16S rRNA gene and tRNA

Leu, Ala, and Ser, two nuclear ribosomal genes (complete 18S rRNA and a fragment of 28S rRNA)

and two nuclear protein-encoding genes (histone H3 and histone H4). Genetic diversity values within

the family are very high and similar to those present within lumbricids. The genus Hormogaster is

polyphyletic and so is Hormogaster pretiosa, stressing the need of a taxonomic revision. Vignysa

could be the ancestral form of the family, although the relations among genera are not clearly

defined, as shown by the incongruence of results obtained among the different analyses.

Hemigastrodrilus seems to be related to the H. elisae complex, and they are both found in the

Atlantic drainage. H. elisae represents one evolutionary lineage with a high genetic diversity and five

cryptic species (Novo et al. 2009) that we propose as a new genus within Hormogastridae. Corsica

and Sardinia seem to have been under two different genetic influences. The species located in

Corsica and Northern Sardinia seem to be more related to Vignysa, whereas H. pretiosa, in the

Southern area of Sardinia, where it was originally described and seems to be restricted to, is more

closely related to some Hormogaster species from Northeast of Spain, where two different lineages

are present. A molecular clock calibration of the tree using the separation of the Sardinian microplate

(at 33 MY) indicated that the diversification of Hormogastridae may be very ancient, starting from 97

to 75 MY ago.

12


5th to 10


Oral - Session 1

Taxonomic chimaera: an overlooked plague exemplified by the common

nightcrawler (Lumbricus terrestris L.)

Porco David

1, James Samuel

2, Decaëns Thibaud

3, Richard Benoit

3, Erséus Christer

4,

Meusnier Isabelle1, Rougerie Rodolphe

1, Hebert Paul D.N.

1

1Biodiversity Institute of Ontario/University of Guelph

2Biodiversity Institute/ Kansas University

3ECODIV/Université de Rouen

4Department of Zoology/ University of Gothenburg

In spite of a critical contribution to global biodiversity, soil organisms have weakly engaged the

attention of taxonomists compared with epigeic taxa and, even in groups intensively studied such as

lumbricid earthworms, the use of DNA barcodes has recently revealed an unsuspected number of

cryptic species often impossible to separate on a morphological basis. In this study, we used

morphology and COI sequences to assess phenotypic and genetic variability among: (1) 181

specimens of the common night crawler (Lumbricus terrestris Linnaeus, 1758) freshly collected in

European and North American populations; (2) three specimens of the neotype series of L. terrestris

collected in Upsala (Sweden) in 1973; (3) one co-type of Lumbricus herculeus (Savigny, 1826) (a

species recently synonymized with L. terrestris) collected in Versailles (France) in 1826; (4)

specimens of five related Lumbricus species. We obtained full-length barcodes from most of the

freshly collected specimens, and a series of smaller fragments (150bp) assembled in a sequence of

480bp for the co-type of L. herculeus. In addition a mini-barcode of 150bp was produced from one of

the three specimens of the L. terrestris neotype series. The fresh specimen sequences highlight the

existence of two different groups of L. terrestris, which are slightly different by morphology (body

size and weight), but have strongly divergent COI sequences (mean distance of 17.6%). A Neighbor-

Joining tree of all sequences indicates that L. terrestris s.l. is paraphyletic with L. festivus the sister

group to the L. terrestris clade containing the L. herculeus co-type specimen sequence. The sequence

from the specimen of the L. terretris neotype series was in the other L. terrestris clade. These results

indicate that L. terrestris as traditionally identified is composed of two cryptic species that have not

been discriminated in the literature. One of these species is the now valid L. herculeus. Based on a

detailed meta-analysis of the scientific literature dedicated to ―L. terrestris‖, we demonstrate that this

taxonomic confusion may dramatically alter the reliability of published data. The attribution to a

single scientific name of characteristics measured on two cryptic species may in fact lead to the

description of a kind of chimerical organism which finally does not correspond to the true properties

of any of the real species.

13


5th to 10


Oral - Session 1

Genetic diversity and structure in European populations of Lumbricus

terrestris (L. 1758) (Lumbricidae) as detected by Amplified Fragment

Length Polymorphism (AFLP)

Potthoff Martin

University of Goettingen

To explore the genetic structure in populations of the anecic earthworm Lumbricus terrestris on a

European scale we investigated populations at 14 locations in Europe. The study was driven by two

main questions: (1) Is genetic diversity of populations decreasing with increasing post glacial

migration distance and/or geographic isolations? and (2) Can we identify preferential distribution

pathways of L. terrestris by comparing the genetic patterns of different populations. An adapted

AFLP method (Amplified Fragment Length Polymorphism) was applied to this species. There was

no clear overall pattern of genetic diversity. However, low values in France and Sweden could be

explained by specific origins or geographical characteristics of sampling locations. The Monmonier

algorithm, the Wombling method and cluster analysis based on different genetic distance measures

identified genetic boundaries in Europe, i. e. regions with a sharp change in genetic information.

These boundaries indicate several regions in Europe with high genetic similarity, probably due to an

origin from the same refuge populations after the Last Glacial Maximum. L. terrestris populations

from the Balkans are connected to Northern Europe, i. e. Sweden and Finland, whereas France and

possibly Italy are more isolated from the rest of Europe. There is strong evidence that L. terrestris

depended on human agricultural activity to spread across Europe and establish new populations. The

pattern of spread of agriculture, during the neolithic revolution, approximately 9000 years ago, can

help to explain today‘s patterns found in the genetic structure of L. terrestris populations. Molecular

methods, like AFLP, are helpful tools to investigate origin, dispersal, genetic diversity and dynamics

of soil organisms which are often difficult to monitor.

14


5th to 10


Oral - Session 1

DNA bar-coding of New Zealand endemic earthworms

Boyer Stephane, Wratten Stephen D.

Bio-Protection Research Centre, Lincoln University, New Zealand

Endemic earthworms from New Zealand were described by Lee in 1959. Since this date, very few

studies have focused on this important group and Lee‘s taxonomic key remains the only resource

available to identify what are thought to be the 170 species endemic to New Zealand. As in most

taxonomic keys, it is mainly based on sexual characteristics that are visible only on adult specimens.

However, the majority of earthworm individuals collected during ecological studies are juveniles and

therefore cannot be identified in this way. This problem is particularly true in communities that

contain many species because juveniles from different species can easily be confounded. As part of

an opencast coal mine restoration project, an inventory of endemic earthworms communities from the

West Coast of New Zealand's South Island was made in various habitats across a 2600 ha area. We

used recognizable taxonomic units (RTUs) to separate juveniles collected in the vicinity of a coal

mine and we performed DNA analyses to check for RTU reliability and to assess the number of

species present. DNA analyses targeting the 16s mitochondrial gene lead to the identification of 17

clades likely to correspond to 17 different species endemic to New Zealand. When adults were

collected, both morphological and DNA analyses were conducted, with results indicating that at least

eight species could be new to science, because adult morphology did not match that of any of the 170

species described by Lee. The discovery of so many new species in a small area (2600 ha) suggests

that the total number of New Zealand endemic earthworm species determined by Lee is likely to have

been greatly underestimated. Although Lee‘s geographical coverage of the country was

comprehensive, New Zealand‘s complex landscape provides many isolated and undisturbed areas

where many earthworm species may remain to be collected and identified. This has important

implications for earthworm conservation and their role in ecological communities.

15


5th to 10


Oral - Session 1

10 years of earthworm molecular phylogenetics: lessons learned

and perspectives

Chang Chih-Han

1, James Samuel

2

1Department of Earth and Planetary Sciences, Johns Hopkins University, Baltimore, MD, USA

2Biodiversity Institute, University of Kansas, Lawrence, Kansas, USA

The upcoming 2011 is the 10th year since the publication of the first molecular phylogenetic study of

earthworms by Jamieson et al. (2002). After that, in the special issues for the 7th and 8th ISEE, two

papers were published by Pop et al. (2003, 2007). With the rapid development on DNA sequencing

technology and analytical tools, however, only a few cases have proved this approach successful. In

the top journals of molecular phylogeny and population genetics, such as Molecular Phylogenetics

and Evolution, Zoologica Scripta and Molecular Ecology, only five full papers focusing on

earthworms have been published, a number much lower than both aquatic oligochaetes and leeches,

and no genus- or family-level revisions have been achieved using molecular tools. On the other hand,

the advances of DNA barcoding seem to be more promising, when used with morphology at the same

time, in (dis)proving taxonomic hypotheses. We briefly reviewed published studies, and compared

them with some of our unpublished data. All the published sequences of the mitochondrial

cytochrome c oxidase subunit 1 (COI) from GenBank were re-examined by reconstructing trees for

different families. We found that molecular phylogenetic trees of earthworms are often characterized

by short internal branches and long external ones, suggesting rapid diversification at different

taxonomic levels, which makes it difficult to achieve a robust tree topology using limited DNA

sequence lengths. We further categorized the commonly used genes into four categories based on

their evolutionary rates, they are (from fast to slowly evolving genes): 1) COI, cytochrome c oxidase

subunit 2 (COII), NADH dehydrogenase subunit 1 (ND1) and internal transcribed spacer (ITS); 2)

12S rRNA and 16S rRNA; 3) 28S rRNA; and 4) 18S rRNA. We then proposed an approach that

analyzes a total of >2,000 bp from different genes at the same time for studying earthworm

systematics. The genes from groups 1 and 2 are suitable for studying intrageneric phylogeny or

related genera, whereas those from groups 2 and 3 are for studying within-family systematics. The

18S rRNA (group 4) is usually used for class- and phylum-level phylogeny and thus is not suitable

for studying earthworms. Finally, for DNA sequences in GenBank, some scientific names of the

sequences are incorrect, which could be from misidentification, misspelling of names and use of

junior synonyms. These data should be carefully used when making taxonomic inferences.

16


5th to 10


Poster – Session 1

A tribute to the enduring status of Dr Ken Lee’s New Zealand earthworm

work

Blakemore Robert1, Boyer Stephane

2

1Nation Museum of Science and Nature, Tokyo

2Bio-Protection Research Centre, Lincoln University, New Zealand

This review is based on the major opus completed over 60 years ago by Ken Lee (1959) as modified

by Blakemore in Lee et al., (2000) and in Glasby et al. (2010) based on the information presented at

the "Species 2000" meeting held Jan. 2000 at Te Papa Museum in Wellington, New Zealand and

finally published in 2010. Lee not only described local species, he also revised all major world

families into a system familiar to students of the classical systematics of Michaelsen (1900) and

Stephenson (1930). In the current checklist Acanthodrilidae, Octochaetidae and Megascolecidae

sensu Blakemore (2000) are all given separate family status, yet much of Dr Lee‘s taxa are still

current and valid. Whereas 193 species were described in Lee (1959), the current list has just over

200 (sub-)spp. The fauna is comparable to adjacent regions, especially Tasmania which at 67,800

Km2 is about one quarter the size of New Zealand (267,000 Km2) yet has 230 known earthworm spp

(Blakemore, 2000). However, as much of the north island of NZ is volcanic and the south island is

partly alpine less area is available for successful earthworm colonization and perhaps a lesser

biodiversity is to be expected. Ken Lee‘s earthworm work was heavily grounded in applied ecology

and soil management. He devised a broad classification of ecological strategies applicable to all

major families of earthworms, rather than later systems that were more applicable to just

Lumbricidae in Europe. Lee‘s (1959) prior ecological classification is restated.

17


5th to 10



An annotated update on the British and Irish earthworm checklist with

new records from Botanic Gardens

Blakemore Robert

National Science Museum,Tokyo, Japan.

All known megadrile species reported from the UK and Eire are presented in an annotated checklist

including the first record from Ireland of Amynthas corticis (Kinberg, 1867) and Pithemera bicincta

(Perrier, 1875) from the Botanic Gardens, Dublin. That some Lumbricidae (except possibly the North

American Bimastos) may have originated in the Middle-East, and been introduced along with

agriculture since the Neolithic, is supported by several species in common with the 34 Levant species

from Pavlicek et al., (2004). Additional reports of megadriles introduced from around the world

being recovered from Gardens, such as at Kew and Dublin, attest to one mechanism of accidental

introduction (and for certain Lumbricidae, Megascolecidae and Eudrilidae there are deliberate

importations for vermi-composting/bait markets). It is unlikely that many of the sub-tropical species

from botanic gardens could readily survive temperate winters outside of the glasshouse, unless there

is substantial climate change. Records from Sims‘ & Gerard‘s (1999) British Isles account were of 48

(sub-species), most of which are commonly found in other countries. The present tally includes

recent reports plus some records marked with ―?‖ giving a new total of ca. 69 taxa.

18


5th to 10



Informatics of Asian earthworms based on ICZN types and COI barcodes

Blakemore Robert

National Science Museum, Tokyo, Japan.

Opportunity to access type material and genetic sequencing has allowed revision and renewal on

neglected parts of the Asian fauna. Current species checklists and specimen DNA profiles are

uploaded and freely available, e.g. on Annelida Resources (www.annelida.net/earthworm/) and

Genbank (www.ncbi.nlm.nih.gov/genbank/) websites. Most recent data will be summarized in this

paper, part of a short series of pragmatic works on Japanese earthworm eco-taxonomy and its value

to primary production. The first earthworms from the Far East were scientifically described by

Kinberg (1867), Horst (1883) and Michaelsen (1891/2) with primary (10?, 100??) types still extant in

European institutions. Re-evaluation of these and other Asian species, some uncharacterized by types

either by initial oversight or later loss, may require neotypification or topotypic avatars. A

combination of specimen availability, online access to primary descriptive literature, standardized

genetic barcoding and rapid data distribution via Internet publication will allow the taxonomic

service to make rapid, continuously updated progress towards resolving nomenclatural uncertainty.

Ancillary considerations are the ―Clarity of vision and courage of purpose‖ (Wheeler 2004:

http://www.jstor.org/pss/4142253) needed from individual taxonomists and natural history museums

to bring about the revolution of classification into the Bio-Info-Tech Age, especially in 2010 – the

UN designated International Year of Biodiversity

http://www.annelida.net/earthworm/

http://www.ncbi.nlm.nih.gov/genbank/

http://www.jstor.org/pss/4142253

19


5th to 10



Abundance and Distribution of Pontodrilus litoralis (Grube, 1855) on the

coasts near Cabo Rojo, P.R.

Boothby Carlo Eduardo, Borges Sonia, Alfaro Mónica

University of Puerto Rico at Mayagüez

The purpose of this investigation was to determine the structure and dynamics of the populations of

the oligochaete Pontodrilus litoralis on the coasts near the Cabo Rojo Lighthouse in Puerto Rico.

During the months of May of 2008 and February 2009, sampling was made at low tide on the sandy

littoral shores of two beaches on each side of the lighthouse. Distances from the tide mark and depth

stratification in sand were taken to account. Environmental factors such as temperature, pH, soil

moisture, organic matter, and surface accumulation of plant debris were registered. Spatial

distribution of Pontodrilus litoralis was determined to be aggregated in clusters. These clusters were

predominant at the high tide mark, where sand was always humid but never completely submerged

under seawater. Organic matter and temperature were determined to be the primary factors in the

distribution dynamics of this species. Sites with the lowest temperatures seemed to have the greatest

number of individuals. Sites with a high accumulation of surface plant debris presented the lowest

temperatures as well as the highest organic matter contents. Sites with clusters of individuals

recorded the most acidic pH due to heightened cell respiration rates where individuals are numerous.

Only a small percentage of the individuals sampled had reached adulthood, indicating a high

reproductive output once the worms enter the reproductive stage but a relatively slow growth to reach

it. A coastal survey of the island was carried out to examine the presence of Pontodrilus litoralis

along different coastal habitats. This resulted in a significant increase in Puerto Rican records of

Pontodrilus litoralis.

20


5th to 10



Ken Lee's specimens revisited


Bio-Protection Research Centre, Lincoln University, New Zealand

Lee‘s morphology-based keys, coupled with modern molecular techniques were used to identify

earthworm species and estimate species diversity in the vicinity of a coal mine in New Zealand's

West Coast. To help species identification and study earthworm phylogeny, we attempted DNA

sequencing on 9 museum specimens collected by Lee in the 1950s. This project aims at putting

together the taxonomic expertise of Ken Lee and modern molecular techniques for a better

understanding of New Zealand earthworm taxonomy and phylogeny. Because museum specimen are

more than 60 years old and have not always been stored in ideal conditions for molecular analyses,

techniques for analyzing ancient DNA were used an attempt to amplify the DNA. A PCR-based

analysis targeting a short internal fragment of the mitochondrial 16s gene was conducted. DNA was

successfully amplified and sequenced from some 60 year-old specimens. Results were compared to

DNA sequences of recently collected earthworms. This study could be the starting point of

systematic DNA barcoding of Lee's museum specimens. Such a project would provide a useful

database for the identification of New Zealand endemic earthworms in the future, especially for

ecological studies where juveniles can represent 90% of the collected individuals.

21


5th to 10



Taxonomic characterization of Lumbricus species (Oligochaeta:

Lumbricidae): comparison of molecular and morphological phylogenies

Briard Charlène1, Qiu Jiang-Ping

2, Pérès Guénola

1, Guernion Muriel

1, Rougé Laurence

1,

Guiller Annie1, Bellido Alain

1, Cluzeau Daniel

1

1University Rennes 1 - UMR CNRS 6553 EcoBio – Station Biologique Paimpont (France)

2Shanghai Jiao Tong University (China)

Taking into account that earthworms play an important function as key-organisms in terrestrial

ecosystems functioning, the present lack of knowledge on phylogenetic relationship between species,

weakens study interpretations applied in ecology, biodiversity and evolution. In light of this, the aim

of our work is to clarify lumbricid taxonomy: the first objective is to test species distinction based on

morphological characters. The second objective is to compare phylogenetic relationships obtained by

morphological and molecular approaches. In this work, we focus on the genus Lumbricus represented

in France by 6 species. First, we use data stored in Lombricien2000 database, representing the

earthworm taxa collected and the morphological characteristics described by Bouché (1972) for each

species. Species life history traits are included among morphological characteristics (e.g. clitellum

and tuberculum position, pores position, Morren‘s gland, body size). Then, we pursue a molecular

approach on individuals sampled in France. Molecular phylogenetic analyses are based on the

sequences of nuclear (18S, 28S) and mitochondrial (16S, COI, COII) gene regions and performed

with maximum likelihood and bayesian inference. Statistical processings are realized on

morphological data to study the contribution of morphological characteristics to separate species

(Multiple Correspondence Analysis) and to define interspecific relationships (Hierarchical Ascendant

Classification and maximum of parsimony). Further statistical analyses are implemented to compare

information from molecular and morphological data to build phylogenetic relationship. Our genetic

results corroborate the taxonomy of the different species studied and confirm that morphological

characters are pertinent to determinate species in this genus. Our molecular phylogenetic results

enable us to propose a hypothesis about the relationship among Lumbricus species. Additional work

of our study should be developed to test our different hypotheses, notably by applying new molecular

markers described in the literature, or going to be developed. A further approach will include

measuring morphological characteristics on earthworm individuals sampled and compared to

molecular analyses to permit a real comparison between the two types of data.

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5th to 10



Spatial Distribution of Primitive Genera in Lumbricidae

Briard Charlène1, Guernion Muriel

1, Rougé Laurence

1, Qiu Jiang-Ping

2,

Cluzeau Daniel1


2Shanghai Jiao Tong University

Earthworm paleogeographic studies are limited by a lack of fossil individuals. However, their limited

species distributions due to a low dispersion, associated to a low number of families, are relevant to

test the geographical history of plates. Indeed, our work is based on the morphological studies

initiated by Bouché (1972) on certain genera of Lumbricidae to explain the evolutionary history of

the North Mediterranean area. According to their morphology and ecology, species belonging to

Prosellodrilus, Zophoscolex and Scherotheca genera are considered as primitive. Several

characteristics states present in Lumbricidae firstly appeared in Prosellodrilus genus (e.g. setae

distribution, diverse bladder types) suggesting that this genus is the most primitive in Lumbricidae

family but the evolutionary meaning remains unclear (Qiu, 1998). Moreover, as Zophoscolex genus,

Prosellodrilus genus contains endogeic species. The genus Scherotheca presents adaptations related

to the anecic mode of life, such as a strong development of capacities to move in soils (e.g. long

body) and to feed on superficial litter (e.g. developed gizzard). Furthermore, this genus seems to

diverge from Prosellodrilus genus. According to these particular features and their presence in the

south of France, the aim of this work is to try to explain the present distribution of these three genera

focusing on history of the North Mediterranean area. We use data stored in Lombricien2000 database

containing sampling sites (around 2000), the morphological characteristics (around 210) described by

Bouché (1972) for each taxon collected and related to each site. We established the cartography of

each genus (and subgenus) using ArcGIS software based on sampling sites coordinates in order to

have a visual idea of species distribution. Statistical processings are realised to study the contribution

of morphological characteristics (e.g. clitellum and tuberculum position, pores position, Morren‘s

gland, typhlosolis, body size) to separate species (Multiple Correspondence Analysis) to interspecific

relationship (Hierarchical Ascendant Classification). Results demonstrate a particular distribution of

species in south of France, structured and associated to a subgenus classification. The link between

species distribution and species phylogeny based on morphological analyses will be presented and

discussed in order to develop phylogeographic hypotheses.

23


5th to 10



Phylogenetic analysis of New Zealand earthworms (Oligochaeta:

Megascolecidae) reveals ancient clades and cryptic taxonomic diversity

Buckley Thomas R

.1, James Samuel

2, Allwood Julia

1,3, Bartlam Scott

4, Howitt Robyn

1, Prada

Diana1

1Landcare Research, Private Bag 92170, Auckland, New Zealand

2University of Kansas

3School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

4Landcare Research, Private Bag 3127, Hamilton, New Zealand

We have constructed the first ever phylogeny for the New Zealand earthworm fauna

(Megascolecinae and Acanthodrilinae) including representatives from other major continental

regions. Bayesian and maximum likelihood phylogenetic trees were constructed from 427 base pairs

from the mitochondrial large subunit (16S) rRNA gene and 661 base pairs from the nuclear large

subunit (28S) rRNA gene. Within the Acanthodrilinae we were able to identify a number of well-

supported clades that were restricted to continental landmasses. Estimates of nodal support for these

major clades were generally high, but relationships among clades were poorly resolved. The

phylogenetic analyses revealed several independent lineages in New Zealand, some of which had a

comparable phylogenetic depth to monophyletic groups sampled from Madagascar, Africa, North

America and Australia. These results are consistent with at least some of these clades having

inhabited New Zealand since rifting from Gondwana in the Late Cretaceous. Within the New

Zealand Acanthodrilinae, major clades tended to be restricted to specific regions of New Zealand,

with the central North Island and Cook Strait representing major biogeographic boundaries. Our field

surveys of New Zealand and subsequent identification has also revealed extensive cryptic taxonomic

diversity with approximately 48 new species sampled in addition to the 199 species recognized by

previous authors. Our results indicate that further survey and taxonomic work is required to establish

a foundation for future biogeographic and ecological research on this vitally important component of

the New Zealand biota.

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5th to 10



Phylogeny of the genus Balanteodrilus (Oligochaeta: Acanthodrilidae)

Casallas Adriana, Fragoso Carlos, González Dolores, Campos Elizabeth

Red de Biodiversidad y Sistemática. Instituto de Ecología A.C. Xalapa, Veracruz, MEXICO

For more than 70 years the genus Balanteodrilus Pickford 1937 remained monotypic, with

Balanteodrilus pearsei as the only described species. B. pearsei is an endogeic polyhumic species

that exhibits variation mainly in size and genital markings, and that is widely distributed in

southeastern Mexico and Belize. Recently, two species were described from the same genus: B.

psamophilus and B. extremus, both of them separated from B. pearsei only by the posterior

displacement of prostatic pores (20 and 21 respectively). In one locality B. psamophilus was found

coexisting with a small morph of B. pearsei, and it was hypothesized that differences in length could

be the result of character displacement or the effect of very sandy soils. Recently, however, more

populations of the small morph were localized in different localities and in one case it coexisted with

large individuals of the same species (B.pearsei). Likewise ecological experiments on reproductive

isolation suggest that the small morph represents a different biological species. In order to clarify if

the three named species (B. pearsei, B. psammophilus and B. extremus) and the small morph of B.

pearsei are valid phylogenetic species, we will conduct a phylogenetic analysis using sequences of

mithocondrial COI (710bp) and 16s (439bp) and nuclear ITS (ca. 600bp) genes of 3 specimens per

location of B. psammophilus, B. extremus and several populations of B. pearsei including the small

morph. DNA sequences will be analyzed using Maximum Parsimony, Maximum Likelihood, and

Bayesian; as outgroups we selected Kaxdrilus silvicola and Lumbricus terrestris. We expect to find

two lineages of evolutionary divergence in the genus Balanteodrilus, one related to size and the other

one to displacement of prostatic pores.

25


5th to 10



Earthworms from Central Mexico: the importance of mountains and

undisturbed forests for native species maintenance

Cervantes Gabriela, Fragoso Carlos, Angeles Antonio

Red de Biodiversidad y Sistemática. Instituto de Ecología A.C. Xalapa, Veracruz, MEXICO As a part of a general project that intends to characterize earthworms in the plateau and mountains of

central Mexico, we sampled earthworms in several, low disturbed sites of the state of Puebla. Sites

included pine, oak and fir forests of several important mountains, as well as some lowland pastures

and tropical forests. Sampling was mainly qualitative, although in some sites 10 quantitative

monoliths were made (50 x 50 cm side and 20 cm depth). Sampling was conducted during the rainy

season of years 2007-2009. We found 20 species, 7 native and 13 exotic. All native species belonged

to Acanthodrilidae, within subfamilies Acanthodrilinae (five species), Benhamiinae (one) and

Octochaetinae (one). Native species were found inhabiting well preserved mountain forests, although

they were always less abundant than exotics. From a biogeographic point of view, native earthworms

characterized the following regions: Neo-volcanic Axis: Protozapotecia sp., Zapotecia sp.,

Dichogaster eiseni and Ramiellona sp.; Gulf of Mexico: Balanteodrilus pearsei and Diplocardia sp.,

and Oriental Sierra Madre: Protozapotecia aquilonalis. The native and new species found in this

study confirm that Mexican mountains still harbor a rich earthworm fauna, which however is highly

threatened by human disturbance and exotic invasions.

26


5th to 10



Regional earthworm DNA barcode database, BOLD system and their

applications: a case study from Taiwán

Chang Chih-Han

1, James Samuel

2, Chen Jiun-Hong

3, Shen Huei-Ping

4,

Szlavecz Katalin1, Chih Wen-Jay

3, Liao Jyun-Ming

3

1Department of Earth and Planetary Sciences, Johns Hopkins University, Baltimore, MD, USA

2Biodiversity Institute, University of Kansas, Lawrence, Kansas, USA

3Department of Life Science, National Taiwan University, Taipei, Taiwan

4Habitat and Ecosystem Division, Taiwan Endemic Species Research Institute, Nantou, Taiwan

The application of DNA barcoding in soil ecology and other disciplines that use earthworms as

model organisms requires a DNA barcode database for the regional fauna. However, to the best of

our knowledge, no such database has been published for any country. In this study, we present results

from Earthworms of Taiwan [EWTW] project, one of the world‘s earthworm DNA barcoding

campaigns registered in the Barcode of Life Data (BOLD) Systems. Our current database comprises

470 sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) from 82 species, including

30 undescribed species and 52 out of the 75 described species from Taiwan. The results support

monophyly of most species. Intraspecific variations were generally high in endemic species but low

in cosmopolitan ones. Levels of sequence variation are appropriate for species separation but not

suitable for interspecific phylogenetic reconstruction. Furthermore, we demonstrate the application of

the Taiwanese earthworm database and the BOLD Systems in three current studies: 1) the

parthenogenesis of Amynthas catenus and Amynthas hohuanmontis; 2) the identification of the first

domicile earthworm sample from Asia (which is also the first record outside the Europe and the

Middle East); and 3) a soil ecotoxicological study using the model animal Eisenia andrei. Finally, we

show that an integrative taxonomic approach adopting both morphology and DNA barcodes may

facilitate the discovery and description of new species.

27


5th to 10



Phylogenetic relationships of the Aporrectodea caliginosa earthworm

complex based on DNA sequences

Domínguez Jorge

1, Ricoy Maigualida

1, Mariño Fuencisla

1, Pérez Losada Marcos

2

1Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, E-36310, Spain

2CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Campus

Agrário de Vairão, 4485-661 Vairão, Portugal

The Aporrectodea caliginosa species complex includes the most abundant earthworms in grasslands

and agricultural ecosystems of the Paleartic region. Historically this complex consisted of the

following taxa: A. caliginosa s.s. Savigny, 1826, A. trapezoides Dugés (1828), A. tuberculata (Eisen,

1874), and A. nocturna Evans (1946). These four taxa are morphologically very similar and difficult

to differentiate because of their morphological variability. Consequently, their taxonomic status and

their phylogenetic relationships have been a matter of discussion for more than a century. To study

these questions, we sequenced the COII (686 bp), 12S (362 bp), 16S (1200 bp), ND1 (917 bp), and

tRNAsAsn- Asp-Val-Leu-Ala-Ser-Leu (402 bp) mitochondrial and 28S (809 bp) nuclear gene

regions for 85 European earthworms from 27 different localities belonging to the A. caliginosa

species complex and four outgroup taxa. DNA sequences were analyzed using maximum parsimony,

maximum likelihood, and Bayesian approaches of phylogenetic inference. The resulting trees were

combined with morphological, ecological, and genomic evidence to test species boundaries (i.e.,

integrative approach). Our molecular analyses showed that A. caliginosa s.s. and A. tuberculata form

a sister clade to A. trapezoides, A. longa, and A. nocturna, which indicates that A. longa is part of the

A. caliginosa species complex. Our analyses also showed the presence of highly divergent lineages

within A. caliginosa, A. trapezoides, and A. longa, suggesting the existence of cryptic diversity

within these taxa.

28


5th to 10



Molecular discrimination of cytochrome c oxidase in Eisenia andrei and

Eisenia fetida

Dvorak Jiri

1, Silerova M.

1, Prochazkova P.

1, Joskova R.

1, Pizl Vaclav

2, Bilej Martin

1

1Institute of Microbiology AS CR, Prague, Czech Republic

2Biology Center AS CR, Ceske Budejovice, Czech Republic

Eisenia andrei and Eisenia fetida are two closely related epigeic earthworm species. Taxonomic

identification of earthworms is often difficult since most of morphological and anatomical

characteristics are variable. These two species were first described as a different morphotypes of E.

fetida according to differences in body pigmentation. Later on they have been considered as

subspecies (E fetida andrei, E. fetida fetida). Therefore we aimed to develop a new approach for

objective molecular determination of these species, namely molecular analysis of gene for

cytochrome c oxidase was performed. Total RNA was isolated and reverse-transcribed using

Superscript II RNase H- Reverse Transcriptase. Resulting cDNA was used for PCR reaction. PCR

products were cloned and the complete sequences of cytochrome c oxidase of both species were

obtained. Cytochrome c oxidase sequence data sets were aligned. Sequence analyses confirmed their

identity as distinct species. Based on these results we have designed sets of specific primers for both

species. The simple PCR reaction is then required for reliable, simple and relatively rapid

discrimination of E. andrei and E. fetida.

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5th to 10



Genetic variability and population genetics in the parthenogenetic

lumbricid Aporrectodea trapezoides (Dugès, 1828)

Fernández Rosa, Novo Marta, Almodóvar Ana, Díaz Cosín Dario J.


Parthenogenesis is a common phenomenon among lumbricids. Until the moment, little attention has

been paid to shedding light into the genetic diversity of this kind of earthworms. This study examines

the genetic variability and population genetics of Aporrectodea trapezoides (Dugès, 1828). All the

studied earthworms were morphologically and anatomically identified as A. trapezoides following

the taxonomic key in Gates (1972), specially taking into account the position of setal genital

tumescence (in segments 9, 10 and 11), the location of tubercula pubertatis (undivided in segments

31 to 33) and male sterility (empty spermathecae and male funnels, both in segments 10 and 11). A

previous phylogenetic analysis (Fernández et al., in prep) showed that this species is clearly divided

in two main lineages: lineage A in Northern Spain and Europe, and lineage B in the Central and

South part of the Iberian Peninsula and other Mediterranean countries. One mitochondrial gene

(cytochrome c oxidase subunit I, 456 bp) and one nuclear gene (histone 3, 311 bp) were chosen to

analyse in depth the genetic variability and population genetics in both lineages. In total, 189 and 79

individuals (Cox I and H3, respectively) collected in 36 localities from 8 different countries were

sequenced. The Cox I data revealed 31 haplotypes, one of them shared by half of the sequenced

individuals. The H3 data revealed 8 haplotypes. In both cases, the haplotypes were exclusive to every

lineage. Both genes showed higher haplotype and nucleotide diversity than expected in a

parthenogenetic species. Genetic divergence between lineage A and B in both genes was very high,

suggesting that the two lineages are cryptic species.

30


5th to 10



Construction of an Eisenia fetida Bacterial Artificial Chromosome (BAC)

library and the genomic analyses of VCP genes

Gamou Shinobu1, Suzuki Kohei

2, Sasaki Takashi

3, Shimizu Nobuyoshi

3

1Kyorin University School of Health Sciences, Tokyo, JAPAN

2STAFF Institute, Tsukuba, JAPAN

3Keio University School of Medicine, Tokyo, JAPAN

Earthworms are established environmental indicators and possess unique biological features;

however the detailed knowledgebase of underlying molecular mechanisms remains sparse. Suzuki et

al.(Gene 362:11, 2005) previously described the genes which are specifically expressed in the gonads

of Eisenia fetida (Savigny, 1826) and isolated two genes, evcp1 and evcp2, with high homology to

mammalian valosine-containing protein (VCP) genes. To define the origin of evcp1 and evcp2 and to

characterize the genomic structure of Eisenia fetida, a bacterial artificial chromosome (BAC) library

was constructed containing in excess of a 5 fold genome coverage spread over 50,000 clones. Two

clones (20E8 and 91F3) were isolated that contain the entire genomic region of evcp1 and evcp2,

respectively. The gene structures including exon-intron junctions were shown to be similar to the

mammalian counterparts, suggesting that the genes have evolved from the same origin. Furthermore,

the entire genomic sequence for 20E8 (122,785bp) and 91F3 (117,704bp) were determined and the

respective GC contents were 41.5% and 41.1%. Genomic informatics analyses revealed that 20E8

contained at least 3 genes, including the malignant T cell amplified sequence 1 (MCTS1), the

RIB43A domain with coiled-coils 2 (RIBC2) and evcp1. 91F3 contained at least 2 genes, a gene with

cyclin L1 (CCNL1) motif and evcp2. Gene structures analyses confirmed that evcp1 and evcp2 genes

occupied approximately 19 kb and 22 kb and consisted of 16 and 17 exons, respectively. Promoter

prediction analysis of each gene revealed the presence of common motifs observed in high

eukaryotes. Repbase analysis identified 49 and 51 microsatellites in 20E8 and 91F3, respectively.

Likewise, many transposons and retroposons were found covering some 6.1% and 6.4% of the

sequences. Among them, tRNA derived short interspersed nucleotide elements (SINE) were

identified, previously characterized in other animals. To date, comprehensive whole genome analyses

have, at large, focused on vertebrates and model organisms. This study provides, for the first time in

Annelida, a stretch of continuous genomic sequence in excess of 100 kb, an imperative source for

future genome assemblies.

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5th to 10



Genomic Sequencing of Eisenia fetida and Lumbricus rubellus BAC clones

containing metallothionein genes

Gamou Shinobu

1, Aimi Yuki

1, Kubo-Murata Makiko

1, Suzuki Kohei

2,

Stürzenbaum Stephen R.3

1Kyorin University School of Health Sciences, Tokyo, JAPAN

2STAFF institute, Tsukuba, JAPAN

3King's College of London, London, UK

In order to characterize the structure of the earthworm genome, bacterial artificial chromosome

(BAC) libraries were constructed from two Lumbricide earthworms, namely Eisenia fetida and

Lumbricus rubellus. Several clones containing the metallothionein (MT) gene were identified from

the libraries via a PCR screening approach. The genomic sequences were determined ―end to end‖ by

shotgun sequencing for four MT containing BAC clones, 318A10 and 472C1 from E. fetida and

13H12 and 6F14 from L. rubellus. 318A10 was 117kbp in length with a GC content of 40.3%. Blastn

confirmed the presence of all 4 exons of the MT gene as previously suggested (Stürzenbaum et al.,

2004. Environ. Sci. Technol. 38 (23): 6283 - 6289). In addition, the GeneScan program identified

that a gene homologous to SLC25A (solute carrier family 25 member 24) is located on the 5‘ side of

the MT gene. The BAC clone 472C1 consisted of 20kbp of 5‘ and 75kbp 3‘ fragment.

Restriction/electrophoresis analysis, combined with in silico analysis, indicated that the two

fragments were separated by a 3kb un-sequenced gap region located in intron 1 of the MT gene.

Unique 500bp repetitive sequences were located in both ends of the gap. Although 472C1 displays a

relatively high homology to 318A10 (approx. 70%), it is suggested that 472C1 and 318A10 represent

independent loci which may have been generated through an evolutionary recent gene conversion.

13H2 from L. rubellus consisted of 103kbp with a 2 kbp spanning 42 bps repeated sequence. Since

the Lumbricus 13H2 and Eisenia 472C1 both contained a similar 500bps inverted repeat regions in

intron 1 of the respective MT gene, it is suggested that they are homologous loci. 6F14 from L.

rubellus was 123kbp long with 3 short gaps; consisting of a 6kbp TA repeat, a 2kbp TAG repeat and

a 500 bp poly G sequence. In 6F14, a gene homologous to ABCA3 (ATP-binding cassette, sub-

family A member 3) was located to the 3‘ side of the MT gene. Since the MT gene in 6F14 was

found to be different in structure from those in other BACs, 6F14 was deemed to be a locus distinct

from 13H2. Ongoing genomic analyses utilizing the BAC libraries are expected to provide not only

the insights into the genomic architecture, but also provide vital information on the evolutionary

relationship between earthworm species.

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Can mitochondrial genes help identify earthworms?

Klarica Jasmin1, Brandstaetter Anita

2, Traugott Michael

1, Juen Anita

1

1University of Innsbruck, Institute of Ecology

2Innsbruck Medical University, Sektion für Genetische Epidemiologie

Developping a reliable and fast method for species identification is an essential tool for studies

dealing with earthworms. However, the lack of morphological characters is the main reason for

identification problems as well as for taxonomic inconsistencies. Could molecular methods possibly

provide a means to overcome these problems? Here, we compared the four most frequently used

mitochondrial gene fragments in earthworm studies – 12S, 16S, COI, and COII – to assess their

suitability for species identification. Earthworm specimens of 14 species collected in different

countries of the northern hemisphere were included. Sequences were analysed using three

phylogenetic methods: (i) Bayesian statistics, (ii) p-distances within species, and (iii) phylogenetic

networks. We found high numbers of haplotypes, deep genetic lineages, and high p-distances in all

four gene fragments of some species. The network based on the 12S sequences was clearly structured

and reflected best the current taxonomic system. Of the four gene fragments analysed, the 12S

seemed to be most suitable for molecular identification of earthworm species. However, due to the

high number of haplotypes found in several species, more sequencing data of more individuals are

needed to detail the picture of the genetic diversity in earthworms. As the results also indicated

cryptic diversity, further investigations are required to clarify the taxonomic situation in Lumbricus

rubellus, Aporrectodea rosea, Octolasion lacteum, and Allolobophora chlorotica.

33


5th to 10



Earthworm communities on Mount Nimba, a West African mountain

Lavelle Patrick1, Omodeo Pietro

2, Somon Silvia

2, Lamotte Maxime

3

1Université Pierre et Marie Curie (IRD-CIAT TSBf Institute)

2Universita di Sienna, Italy

3Museum national d'Histoire Naturelle

Mt Nimba is a 1752 mountain m located in Western Africa. Fourty seven earthworm species have

been collected on a surface of a few hundred Km² which makes this site the richest ever found until

now. A majority of species was found in only one (21) or two (10) of the 87 sites sampled while only

two species, Dichogaster leroyi (30 sites) and D. lamottei (40 sites) were the most common species

and the Eudrilidae Stuhlmannia zielae the only species with a rather broad regional distribution.

Species richness in 50 cm x 50 cm x 30 cm depth samples varied from 1 to 9 and density was up to

1200 ind m² with an average of 250. Correspondance analysis based on species biological traits

showed a significant segregation according to altitude (p < 0.05). Eleven species were only found in

piedmont areas (< 700m elevation) while 10 were restricted to the grasslands and shrub vegetation of

the higher part of the mountain (> 1300m) and 12 were found at all altitudinal stages. No significant

effect of vegetation type on communities was observed. Species from the piedmont area were

generally large sized, unpigmented with well developed typhlosolis indicating geophagous feeding

regimes. On the opposite, species only found at high elevations presented a set of characters

(pigmented, relatively small with well developed setae and reduced typhlosolis) that suggest litter

feeding habits. Species found at all altitudes presented intermediate characters. This study is a clear

example of the high speciation rates that may occur in earthworms when diverse environment

conditions are found in a relatively small region.

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Cryptic speciation within Hormogaster elisae revealed by molecular data

Novo Marta, Fernández Rosa, Trigo Dolores, Almodóvar Ana, Díaz Cosín Dario J.


Hormogaster elisae (Hormogastridae) is an endogeic earthworm endemic to the central area of the

Iberian Peninsula. This species can be very abundant in sandy and poor organic matter soils, with

strong erosion and summer aridity. Climate change will presumably favour the extension of this kind

of soils, where Hormogaster species and specifically H. elisae could be the only earthworms capable

of thriving. Because of that, their ecological importance could become crucial and hence, it is

necessary to look into their genetic variability in order to develop conservation and management

programmes. One nuclear (28S rRNA) and two mitochondrial (COI and 16S rRNA) genes were

sequenced in individuals from 16 populations of the central Iberian Peninsula where soil properties

(texture, carbon, nitrogen and pH) were measured. The morphology of the specimens was carefully

studied. The overall genetic diversity was very high and suggested the occurrence of several cryptic

species within this region. For instance, the barcoding gene COI showed 80 haplotypes (exclusive to

localities) out of 202 individuals and exhibited values of genetic divergence among populations up to

22%. Moreover, the studied genes agreed to cluster the same evolutionary lineages, indicating that

different phylogenetic species could be defined. Nevertheless, a morphological constancy was found

that led to the conclusion that all studied individuals belonged to the morphospecies H. elisae. These

results could indicate discordant patterns of morphological and molecular evolution or a

morphological stasis, which seems to be common in non-visual invertebrates and lead to cryptic

speciation. The unique morphological distinction among the studied populations was the size of

specimens, but its differences were not related to the genetic divergence. C/N exhibited a positive

correlation with the weight of the collected specimens. Colonization events seem to have been the

main cause of the current scenario as shown by a pattern of isolation by distance. Furthermore, a

positive although weak correlation was also found between some soil properties (coarse sand and

total loam content) and genetic distances, which indicates that these populations could have been

shaped genetically, but not morphologically, by the environment.

35


5th to 10



Malagasy earthworms

Razafindrakoto Malalatiana1, Blanchart Eric

2, Csuzdi Csaba

3

1University of Antananarivo/IRD

2IRD

3Museum of Natural History, Budapest

Madagascar and surrounding islands in the Indian Ocean constitute one of the 25 biodiversity

hotspots in the World. These islands are characterized by very important endemisms and many new

species are identified every year. However earthworms of Madagascar are poorly studied. The last

scientific taxonomic report on a Malagasy earthworm dates from 1931 and was published by

Michaelsen. Only 35 species were described at this date. A survey on earthworms and other soil

engineers started in 2008. The objectives are to study the taxonomy, ecology and distribution of

Malagasy earthworms. For 2 years, earthworms were sampled in different pedoclimatic areas, in

different land uses. To date, many species have been sampled and are being identified. They belong

to 6 different families: Megascolecidae, Kynotidae, Acanthodrilidae, Eudrilidae, Ocnerodrilidae,

Glossoscolecidae. Among the species collected so far 8 are new to science and most of them belong

to the endemic family Kynotidae (only 16 species of this family are known). The large size of

Madagascar could explain a large number of earthworm species, and especially of endemic species.

Other than the Kynotidae family, we found an endemic genus Howascolex (Octochaetidae) and three

endemic species belonging in the South-African genus Eodriloides (Acanthodrilidae). The present

work describes the actual knowledge on taxonomy and distribution of Malagasy earthworms.

36


5th to 10



FISH mapping of rDNA and histone gene clusters in earthworms

Sánchez-Conde O., Pérez-García C., Briones Maria J.I. Pasantes J.J.

Universidad de Vigo

The phylogenetic relationships within earthworms have usually relied on morphological characters

which show some limited value due to their enormous variability, even among individuals of the

same species, and have led to several taxonomical ambiguities which remain unresolved. Although

the recent use of molecular tools in earthworm phylogeny has contributed to clarify some

controversial taxonomical aspects, it has also posed new questions. For example, in the case of

certain species included in Dendrobaena, Eisenia and Aporrectodea the resolution provided by the

molecular markers is still quite limited. Therefore, in order to provide a better understanding of the

evolutionary relationships within Lumbricidae here we provide an analysis of the distribution of the

major rRNA, minor rRNA and histone gene families in several species of earthworms. Chromosome

preparations were obtained from seminal vesicles after hypotonic treatment and fixation with

ethanol/acetic acid. Chromosome counts were carried out and karyotypes constructed. The presence

of CG- and/or AT-rich chromosome regions was analysed and the ribosomal and histone gene

families mapped by means of fluorescent in situ hybridization (FISH) using species specific H3, 28S

rRNA and 5S rRNA gene probes generated by PCR.

37


5th to 10



Species richness and density of earthworms in grasslands of western Uttar

Pradesh, India

Singh Satyendra M., Prakash Om

Vermiculture and Environmental Research Laboratory, Department of Animal Science, Mahatma Jyotiba Phule

Rohilkhand University, Bareilly-243006 (India)

Earthworm species richness and their density reflects the soil quality of an ecosystem, which was

currently unknown in grasslands of Uttar Pradesh. The study was conducted in five regions of

western Uttar Pradesh, India, from years 2006 to 2009, with the aim of quantifying species richness

and density of earthworms in different seasons. Three samples were taken, from each region in every

season, using a metal quadrat of the size 25 x 25 x 20 cm. Worms were identified by observing

external and internal morphological body characteristics and their density was quantified in terms of

their number/m². Three species of Megascolecidae viz., Metaphire posthuma (Vaillant), Lampito

mauritii (Kinberg, 1867) and Perionyx excavatus (Perrier, 1872) and four species of Octochaetidae,

Eutyphoeus waltoni (Michaelsen, 1907), E. gigas (Stephenson, 1917), E. orientalis (Beddard, 1883)

and E. pharpingianus (Michaelsen, 1907) were identified and recorded. Species richness and density

of worms were highest in Moradabad during rainy season (July to October), whereas density was

minimal in Saharanpur during the winter months from November to February. Species richness of

worms was found to be the lowest in Meerut region during winter and summer months and in

Saharanpur only during summers (March to June). M. posthuma was abundantly found in all the

regions round the year. In addition, L. mauritii and E. waltoni have been reported from all the regions

only during the rainy season. P. excavatus was next in occurrence pattern, as it was recorded from all

the regions except from Meerut. Occurrence of E. orientalis, E. gigas and E. pharpingianus was

recorded rare. Influence of seasons and climatic conditions in the variation of species richness and

density of earthworms are discussed.

38


5th to 10



Geographical effect on genetic diversity and molecular Evolution of

Eisenia fetida in Northern China

Sun Qian, Zhang Yufeng, Sun Zhenjun

China Agriculture University

Earthworm meta-population is a biogeocenose strongly influenced by fragmentized landscapes.

Based on field investigation conducted on summer 2009, in Shandong Province and Liaoning

Province, China, this study took DNA sequences of 16S rDNA and COI as molecular markers and

analyzed genetic diversity of Eisenia fetida sampled from 4 islands, 2 peninsulas and 6 mainland

localities. Result shows: i) Different populations of E. fetida are divided into 4 clusters, which

correspond to populations of Shandong island, Liaoning island, Shandong mainland (including

peninsula) and Liaoning and ii) Genetic diversity of island populations is richer than mainland

populations. This indicates that seawater, as a kind of geographic barrier, accelerated intraspecific

genetic divergence in E. fetida.

39


5th to 10



Diversity and distribution of earthworm communities in easternmost

Canary Islands

Talavera José Antonio

Universidad de La Laguna, Tenerife, España

Urban and tourist development, still in continual expansion, are probably leading to the

disappearance of significant populations of earthworms in the easternmost Canary Islands, up to now

little investigated. Along with this, the lack of references in the literature and the evident fragility of

the local ecosystems support the urgent need for a study that provides reliable data on the

biodiversity and spatial distribution of the lumbricofauna. Up to now this includes 15 taxa belonging

to Lumbricidae (60 %), Acanthodrilidae (26.66 %), Megascolecidae (6.66%) and Ocnerodrilidae

(6.66 %). Our collection efforts were centered on a minimum of 70 selected sites (with their

respective geographical coordinates and elevation data) to include at least the more representative

biotopes: sectors of relictual native vegetation, tree plantations, agricultural land, semiaquatic

habitats, grassland with dispersed palms, recreational areas (parks, gardens), organic matter

dumpsites, watercourse beds and port areas. Earthworms were collected by digging moist blocks of

soil, pouring on dilute formalin and hand-sorting; identification of the specimens was made with the

aid of a previously compiled taxonomic key. Initially the results reveal that the ubiquitous

Pontodrilus litoralis have a non-random spatial distribution and variable abundances; it was found in

supralittoral pools and ravine mouths, presently disturbed or in danger of disappearance due to the

severe environmental damage. The African Ocnerodrilus occidentalis occurs in semiaquatic habitats

of the basal belt facing southeast, while the exotic invasives Dichogaster affinis, Dichogaster bolaui

and Microscolex phosphoreus have low-density populations spread over anthropic sunny sites, below

100 m a.s.l. Other species are mainly palaearctic peregrines found in diverse microhabitats, from the

coast to mid-alttitude areas and even mountainsides; among them Aporrectodea rosea bimastoides

and Aporrectodea trapezoides show the highest ecological tolerance, having a wide distribution and

average population density. We analyse the invasive front of some species and the different

introduction-dispersal pathways, particularly anthropic (human settlements associated with

agrocommercial activities), biological (vertebrate transport-vectors and exotic plants) and natural

routes (by sea). Our conclusion is that these dispersion patterns have to a large extent determined the

present-day composition of the lumbricofauna, and that together they might provide clues for

understanding the history of the biota of the Canary Islands. In particular, the case of P. litoralis is

discussed, considering that its presence should not be related exclusively or necessarily with human

interference. Other results point out that: i) an increase of more than 50% in the total species range,

ii) Fuerteventura island is more diverse in terms of species richness.

40


5th to 10



Biodiversity of terrestrial earthworms (Annelida: Oligochaeta) in

Shandong peninsula and Liaodong peninsula, China

Zhang Yufeng, Sun Qian, Zhang Donghui, Yao Lan, Xiong Hui


A peninsula is a kind of ecotone connecting land and an island with different characteristics, which is

suspected are also reflected in earthworm biodiversity. A recent survey of earthworm biodiversity

was conducted in China Shandong and Liaodong peninsulas in 2009, which are connected with

terrestrial and Korea Peninsula, respectively. In this paper, we report up-to-date checklists of the

terrestrial earthworms in Shandong peninsula (SD) and Liaodong peninsula (LD). All materials were

collected either by digging and hand-sorting in farmlands, mountains and shoreside of ponds or water

reservoirs, or by searching ground litter, mosses and rotting logs. Earthworms were killed in 70%

ethanol, and then preserved in 95% ethanol for further research. Meanwhile, a part of them were

transferred to 10% formalin for fixation as voucher specimens. Fourteen species are known in SD,

mostly of the Megascolecidae (43% of total) and Lumbricidae (36% of total). But seventeen species

are recorded in LD, mainly within Lumbricidae (47% of total) and Megascolecidae (35% of total).

Drawida spp. and Amynthas spp. are widespread in Dandong, a locality of LD. Eisenia fetida

(Savigny, 1826) was intensely harvested for the vermiculture industry. Considering the importance of

earthworm activity for soil function, and the existence of two new species to be described from LD,

further sampling efforts and taxonomic training of Chinese scientists are urgently needed.

41


5th to 10



Earthworm biodiversity and paleogeography in Hainan Island, China

Zhao Qi

1, Briard Charlène

2, Qiu Jiang-Ping

1, Jiang Jibao

1, Sun Jing

1,

Guernion Muriel2, Pérès Guénola

2, Cluzeau Daniel

2

1Shanghai Jiao Tong University


Earthworms can not readily cross seas, rivers, or mountains; therefore, the modes which make their

distribution restricted can be very valuable in testifying the geographical history of the world. For

example, the paleogeography of Europe can be dovetailed well with the phylogeny of Lumbricidae

(Qiu and Bouche, 1998). However, there is no similar research on Megascolecidae while they are

also widely distributed in many places, especially in Asia and Australia. Hainan Island is the second

largest island of the coast of China. According to the geological history, ingression and regression of

the sea occurred several times around this island, followed by a large coastal plain which enabled

flora and fauna to migrate back and forth from the mainland to Hainan Island (Su 2004). Finally,

Hainan Island was separated from the mainland again in Holocene and became as present (Xing,

1995). In 1938, Chen firstly investigated earthworms in Hainan Island and reported 32 new species

(Chen, 1938), followed by Zhong and Quan who reported another 3 new species (Zhong, 1989;

Quan, 1985). However, after that, no research continued in Hainan. In light of these, we supposed

that there will be maybe more new species in Hainan Island. Moreover we hypothesized that the

phylogeny of Hainan earthworms might support the paleogeographic hypotheses of Hainan Island,

even of China and Asia. In order to confirm our hypothesis, the following methods were applied in

our study: (1) We investigated earthworm biodiversity in Hainan in 2006 and 2009 separately, and

identified them by external and internal morphological characteristics, for example, length, segments,

spermatheca, and so on; (2) Molecular methods (COI, AFLP, RAPD as makers) were used to study

the phylogeny of earthworms; (3) Correspondence factor analysis, Neighbour-joining and Bayesian

inference methods were then applied to analyze the data obtained in step 2. Based on anatomical

characteristics, the earthworm biodiversity in Hainan Island was clarified, and ended in 42

earthworm species, including 10 species which we discovered; 38 species belong to Megascolecidae.

Results were compared to those from molecular approaches and discussed. Furthermore, those results

were used to explain the phylogeny of Megascolecidae in Hainan Island and dovetail the

paleogeography of Hainan Island, even with that of Asia.

42


5th to 10


Session 2

Earthworm Immunology and Physiology

43


5th to 10



Earthworm Innate Immunity in the Genomic Era: The Search for the

Elusive Recognition Receptors

Francis Joe W.1, Cooper Edwin L.

2

1 The Master's College, USA

2 University of California Los Angeles, USA

The innate immune system is the fundamental immune system of all metazoans and metaphytes. It

performs the critical function of pathogen recognition; without it, the vertebrate adaptive response is

severely handicapped. The elucidation of mechanisms of immunity in invertebrates has contributed

much to our understanding of the innate immune system. Indeed the establishment of the discipline

of Immunology was inspired in part by observations of the invertebrate innate immune system. Much

of the work in this area has concentrated on the ―response‖ arm of the innate immune system

corresponding with identification of humoral factors and phagocytes involved in pathogen clearance.

Evidence for the ―recognition‖ function of the innate system and its critical importance has only

recently been established in vertebrates yet it has been recognized as important in invertebrates for

centuries. The identification and characterization of recognition molecules has, however, lagged

behind discovery of the immune response components. Recently, comparative genomics has greatly

aided in the unveiling of pattern recognition receptors and their corresponding signaling pathways in

several invertebrates. For instance, the immune receptors Toll and Nod and some of their signaling

molecules have been detected in representatives of deuterostome invertebrate groups. Innate immune

receptors have also been detected in protostomes, however, only two protostome models of

invertebrate immunity, namely Drosophila and C.elegans, have been examined, in depth, to reveal

both gene identity and receptor function of innate immune system components. Curiously, these

studies show that, the major role of Toll, the most widely studied pattern-recognition receptor, may

not necessarily involve immune function but instead may play major roles in development and

behavior. Recently, Toll has been discovered in the genetic sequences of the annelids Helobdella and

Platyneries. The initial analysis of earthworms using anti-Toll antibodies yielded few results; Toll-

like receptor 2 and a flagellin binding protein were detected on certain cells in Eisenia. In contrast,

preliminary data from analysis of newly partially sequenced genome of L. rubellus shows that Toll

receptors and other innate immune receptors and their corresponding signaling pathways may be

abundant in earthworms affirming that the earthworm immune system is an important model of the

innate immune system.

44


5th to 10


Oral – Session 2

A model of innate immunity in Eisenia andrei earthworms

Bilej Martin 1, Prochazkova P.

1, Joskova R.

1, Silerova M.

1, Dvorak Jiri

1, Skanta Frantisek

1,

Trefilova T. 1, Rossmann P.

1, Kauschke E.

2

1 Institute of Microbiology AS CR, Prague, Czech Republic

2 Justus.Liebig University, Giessen, Germany.

Eisenia andrei, like other invertebrates, do not possess anticipatory immune reactions based on the

presence of lymphocytes and antibodies but they rely on the innate mechanisms that allow them to

survive in often very hostile environments. In the last four decades important cellular and humOral –

Session 2 pathways have been described and numerous biologically active compounds have been

characterized and often cloned: namely, (i) the pattern recognition molecule CCF that binds to

microbe-associated molecular patterns of Gram-negative and Gram-positive bacteria as well as fungi

and triggers prophenoloxidase activating cascade, (ii) important antibacterial proteins fetidin and

lysenin, and (iii) lysozyme provide a useful tool for description of complex mechanisms. We will

present a model of an antimicrobial pathway that begins with recognition of microbe-associated

molecular patterns by CCF in the gut, followed by differentiation of coelomocyte precursors in the

mesenchymal lining. This results in the release of free effector coelomocytes and expression of

immunologically active molecules – fetidin, lysenin, and lysozyme. Simultaneously, CCF triggers

prophenoloxidase cascade that leads to the production of melanin with antibiotic properties.

Detection of increased microbial pressure by pattern recognition CCF in the gut allows timely

mobilization of immune mechanisms to prevent massive uncontrolled overgrowth of microorganisms

in the coelomic cavity and maintenance of homeostasis.

45


5th to 10


Oral – Session 2

Exposure of coelomocytes from Eisenia hortensis to oxidizing agents and

heavy metals: A study examining effects on phagocytosis, apoptosis and

ROS production

Fuller-Espie Sheryl L., Nacarelli Thimoty, Blake Erin L., Bearoff Frank M.,

Minutillo Madeleine M.

Cabrini College, Radnor, Pennsylvania, U.S.A

The first part of this investigation analyzed the effect of exogenous hydrogen peroxide (H2O2) on

phagocytic function and apoptosis in coelomocytes from Eisenia hortensis. Treating coelomocytes

with H2O2 (0.26 to 8.4 mM) evoked a significant increase in phagocytosis for one or more of the

concentrations of H2O2 employed in 67% of cases. Using annexin V-FITC we show that H2O2

induced apoptosis of coelomocytes in vitro. We found that 100% of viable coelomocyte populations

exhibited significant increases in phosphatidylserine translocation for one or more of the

concentrations of H2O2 tested (8.4 to 67.6 mM). Using a fluorescent inhibitor of caspases (FLICA),

we also revealed the presence of activated caspases observing increased caspase activity in 67% of

viable coelomocyte populations treated with 33.8mM H2O2, and in 100% of cases treated with 67.6

mM H2O2. Agarose gel electrophoresis and the TUNEL assay confirmed DNA fragmentation in

samples treated with 16.9 and 33.8 mM H2O2. Thus, exogenous H2O2 facilitated phagocytosis and

promoted oxidative-stress-induced apoptosis, two cellular functions that may play an important

function in regulating innate immune responses in E. hortensis. The second part of this study

examined the effect of heavy metals on the production of reactive oxygen species (ROS). Oxidation

of the fluorogenic substrate dihydrorhodamine 123 was monitored for the detection of ROS in

coelomocytes treated in vitro with copper chloride, cadmium chloride or zinc chloride. Our results

indicate that copper and cadmium induced a statistically significant, dose-dependent response with

ROS production increasing in 40-80% of earthworms treated with copper, and 30-100% of

earthworms treated with cadmium at concentrations ranging from 16 – 250 mcM, respectively. Zinc

induced an elevated ROS production in only 10% of earthworms treated in this range. This method

could be easily adapted for environmental studies investigating contaminated soils using ROS

production in earthworms as an indicator of pollutants.

46


5th to 10


Oral – Session 2

Riboflavin as chemoattractant for earthworm coelomocytes and

mammalian immunocompetent cells

Mazur Agnieszka Irena

1, Morgan John

2, Plytycz Barbara

1

1 Institute of Zoology, Jagiellonian University, Krakow, Poland,

2 Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK

Riboflavin plays an important role in the immunity of plants and animals (as vitamin B2 in the latter)

and in quorum sensing of bacteria. We have shown that riboflavin is stored in chloragocytes attached

to alimentary tissues and in chloragocyte-derived eleocytes freely floating in the coelomic cavity. It

is germane that eleocytes are functionally involved in the formation of multicellular bodies

encapsulating soil-derived pathogens. We may assume that riboflavin modulates the balance between

pathogens and the earthworm innate immune system. Putatively, upon microbial stimulation active

riboflavin metabolites support effectors of immunity, or riboflavin itself is released from

chloragocytes/eleocytes intracellular stores as a chemoattractant for immune-competent cells, i.e.

amoebocytes and eleocytes with phagocytic and bactericidal properties. The aim of the present

investigation was to check if riboflavin acts as a chemoattractant for immune-competent cells from

phylogenetically distantly related taxa, namely earthworm coelomocytes, mammalian leukocytes, and

mammalian cell lines. Earthworm coleomocytes were retrieved by electric shock from Lumbricus

terrestris, Aporrectodea caliginosa and Eisenia andrei. Murine leukocytes were lavaged from the

bone marrow of adult Swiss males. Human monocyte U937 cells, murine monocyte/macrophage

RAW 264.7 cells, and murine fibroblast L-929 cells were cultured in RPMI medium. Tests were

performed in a 48-well chemotaxis chamber. Triplicate wells in the lower part of the chamber were

filled with various concentrations (10-8, 10-6, and 10-4 M) of riboflavin as a putative

chemoattractant. RPMI medium served as a negative control, while zymosan-activated murine serum

(ZAS) or fMLP (chemoattractant of bacterial origin) served as positive controls. Wells of the upper

part of the chambers were filled with cell suspensions. Statistical analysis of the numbers of cells

migrating through polycarbonate filters was performed by ANOVA with post-hoc Tukey‘s test. As

expected, ZAS and fMLP were efficient chemoattractants for all immune-competent cells and L-929

fibroblasts. Riboflavin acted as an efficient chemoattractant for all cell types involved in immune

functions but not for L-929 fibroblast cell line. In conclusion, chemoattractant activity of riboflavin

may be one of mechanisms underpinning its immunomodulatory functions. Acknowledgements:

Supported by DS/BiNoZ/IZ/773/ZIE and N N303 089834.

47


5th to 10


Oral – Session 2

A two-dimensional electrophoresis reference map of the earthworm

Eisenia fetida

Wang Xing

1, Li Chang, Sun Zhenjun, Sun Qian

1

1 China Agriculture University, China

2 College of Life Science, Huaibei Normal University, China

The molecular mechanisms underlying innate immunity in the earthworm E. fetida remain unclear.

For the recognition of innate immunity in the earthworm E. fetida, a detailed knowledge of this

proteome is a prerequisite. The absence of a high-resolution E. fetida proteome map prompted us to

determine E. fetida protein spots that can be visualized on 2-D protein gels. Here, we present a

preliminary description of the whole earthworm E. fetida proteome. A highly detailed 2-DE map of

the E. fetida proteome was established and approximately 1 500 protein spots were detected from the

earthworm sample when applying a 500 µg protein 2-DE in the pH range 3.0-10.0. We present a 2-

DE proteome map of E. fetida, identifying 76 different proteins by MALDI-TOF/TOF MS analysis.

These identified proteins, including Hsp90, fibrinolytic protease 0, gelsolin-like protein, lombricine

kinase, coelomic cytolytic factor1 (CCF 1), MnSOD, triosephosphate isomerase, extracellular globin-

4, lysenin, and intermediate filament protein, glyceraldehyde-3-phosphate dehydrogenase and et al.,

are involved in several processes, including transcription, translation, the tricarboxylic acid cycle, the

cellular amino acid metabolic process, protein amino acid phosphorylation, glycolysis, and the

glucose metabolic process. It will be useful to investigate proteomics in earthworms, which has been

rarely reported, despite of their importance in earthworm biology.

48


5th to 10


Oral – Session 2

Antimicrobial activity of Lampito mauritii (kinberg) extract and its

probable mode of action

Balamurugan M. 1, Parthasarathi K.

1, Ranganathan L.S.

1, Cooper Edwin L.

2

1 Annamalai University, India 2 University of California, USA

Studies were made on the antibacterial and antifungal activity of earthworm extract against eight

pathogenic bacteria and eight fungi. Earthworm extract exhibited antibacterial activity against four

Gram-negative bacteria- Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and

Vibrio cholerae and four Gram-positive bacteria- Staphylococcus aureus, Streptococcus pneumoniae,

Staphylococcus epidermidis and Bacillus subtilis. Earthworm extract also exhibited antifungal

activity against four yeast type fungi- Candida albicans, Candida tropicalis, Candida krusei and

Candida parapsilosis, one dermatophytic fungus- Tricophyton mentagrophytes and three mould type

fungi- Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus. These results suggest the

broad spectrum nature of antimicrobial activity of L. .mauritii extract. The extract of L. mauritii- a

compound seems to be composed of low molecular weight proteins (29 kD, 43 kD and 66 kD)

amongst the other active principles such as phenolic substances and humic substances found in

tissues of the L. mauritii and known to be antibacterial and antifungal.

49


5th to 10


Oral – Session 2

Vermipharmaceuticals isolated from earthworms

Fang Weilin1, Wang Chong

2

1 Bocom Pharmaceuticals Ltd., China 2 China Agriculture University, China

The earthworm is one of the typical saprophagous organisms, which was successfully used to convert

animal waste into biomass. Earthworms contribute internationally by vermicomposting. The

pharmaceuticals from earthworm, as green biomedicine, have developed fast in recent years.

Earthworms have become an international medicine, even though the original utilization of

earthworm as traditional medicine has been known for thousands of years. With the development of

science, scientists found the medical value of earthworms related to many chemical components.

Here we describe vermipharmaceuticals isolated from earthworms, including: fibrinolytic enzymes,

lumbrokinase, collagenase, superoxide dismutase, cholinesterase, catalases, glycosidases,

metallothionein, calmodulin-binding protein, proteins with proliferation improving activity, lysenin,

eiseniapore, antitumor protein, glycoprotein extract, gut mobility regulation peptide, antibacterial

peptide, carbamidine, lumbrin, lumbrofobrim, terrestrolumbrolysin, purin, vitamin B, tyrosine,

succinic acid, lauric acid, unsaturated fatty acid, and so on. This report mainly summarizes fatty

acids, enzyme systems, proteins and peptides.

50


5th to 10


Oral – Session 2

Mitogenicity of earthworm lectins

Nair Preetha, Selvakumar Sharanya, Kale Radha D.

Mount Carmel College, India

Lectins are non-catalytic proteins or glycoproteins that are capable of recognizing and binding

reversibly to specific saccharide moieties of glycoconjugates. Lectins have a variety of biological

properties including antifungal, antiproliferative or mitogenic and hemagglutinating activities. The

ability of lectins to detect subtle variations in carbohydrate structures on the surface of cells and

tissues has made them a paradigm for protein-carbohydrate recognition. Recognition of cell-surface

carbohydrates by lectins has broad implications in important biological processes. Earlier lectins

were thought to be only of plant origin but later their presence was shown even in animal systems.

Animal lectins fulfill a variety of functions. Many could be considered in general terms to be

recognition of molecules within the immune system. More specifically, lectins have been implicated

in direct first-line defense against pathogens, cell trafficking, immune regulation and prevention of

autoimmunity. Invertebrate lectins have highly developed discriminative ability of the clearance

mechanisms. Their binding to non-self materials is based on the presence of carbohydrate-specific

recognition. Lectins or agglutinins are known to occur in the serum of a variety of invertebrates..

Lectins from Earthworm Eudrilus eugeniae was isolated by a lactose-specific extraction in the

absence of detergent and metal ion. EDTA-MEPBS buffer was used for the partial purification of

lectin from whole earthworm. After quantifying the protein content in the lectin sample, carbohydrate

specificity was determined by DNS method using different sugars. The effect of various saccharides

on lectin extraction was investigated and the lectin isolated was found to be galactose binding

protein. The mitogenicity of earthworm lectin on vertebrate cells (liver cells) and invertebrate cells

(coelomocytes and muscle cells from earthworm) were assessed. On addition of lectin; liver cells and

earthworm muscle cells showed increased proliferation and coelomocytes exhibited decrease in cell

number. Antimitogenicity of earthworm lectin on cells of the same species was a novel finding,

therefore by the same isolation method lectin was isolated from coelomic fluid alone. The lectin

showed galactose specificity and showed mitogenicity in coelomocytes. To confirm the results, lectin

was isolated from cultured earthworm muscle cells, fluid from outer region of earthworm muscle

wall, and fresh vermicompost. Carbohydrate specifity and mitogenicity were found to vary. Lectin

from cultured earthworm muscle cells and fluid from outer region of earthworm muscle wall were

found to be glucose binding and has no mitogenic effect on muscle cells where as lectin from fresh

vermocompost was found to be galactose binding. Since lectin isolated from coelomic fluid and

muscular wall cells of earthworm are of different carbohydrate specificity and of different mitogenic

effect. The results confirm that the synergistic effect of lectins of different specificity produced by

different cells is responsible for the characteristic immune response exhibited by earthworm.

51


5th to 10


Oral – Session 2

Oxidative stress caused by UV radiation or heavy metal exposure in

earthworms

Chen Jiun-Hong, Chen Po-Chen, Wu Ya-Fan, Chuang Shu-Chun

Dept. Life Science, Institute of Zoology, National Taiwan University

It has been demonstrated that either UV radiation or heavy metal can cause severe damage in several

earthworm species. UV, especially UV-B which increasingly reaches the earth surface in the past

decades due to the breakage of the ozone layer in the atmosphere, can impair the earthworm skin and

muscle, or affect the earthworm crawling behavior. Cadmium, one of the major heavy metal

pollutants, could be lethal when it is over bio-accumulated into an earthworm. The pathological

responses of UV radiation or heavy metal on earthworms have been described and compared to show

differences. Generally, the damage effect of UV-B results from oxidative stress which includes

reactive oxygen species (ROS) generation. ROS is highly reactive, and can result in significant

damage to cell structures or lipid peroxidation due to the presence of unpaired valence shell

electrons. Surprisingly, in our lab, it was recently found that the impairments of heavy metal on

earthworms also causes oxidative stress. The antioxidant studies have further demonstrated that the

damage effects from either UV radiation or heavy metal exposure in earthworms could be

significantly reduced when antioxidant such as Vitamin C was present.

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5th to 10


Oral – Session 2

Metal mapping and ligand-speciation across the alimentary epithelia of

earthworms native to metalliferous field soils: correlative electron

microprobe and synchrotron microbeam X-ray analyses on frozen sections

Morgan John1, O‘Reilly M.

1, Winters C.

1, Fisher P.

1, Charnock J.M.

2, Kille Peter

1,

Andre Jaqne 1, Turner M.

1, Bennett A.

3

1 Cardiff University, Cardiff, Wales, UK

2 University of Manchester, UK 3 SRS Daresbury Laboratory, UK

The capacity of earthworms to bioaccumulate and resist the effects of soil-borne metal contaminants

is exceptional, with recorded Pb and Zn dry mass concentrations in whole worms from certain

metalliferous soils occasionally exceeding 10,000 µg/g. Only a small fraction of the equilibrated

metal body-burden of earthworm field populations with multi-generation exposure histories can

possibly be bioreactive. Thus, defining the compartmentation and ligand-binding speciation of metals

in earthworms is as important as it is technically elusive. The main aim of the present study was to

use a combination of physic-chemical techniques, electron probe X-ray microanalysis in the

transmission and scanning electron microscopes, together with prototype high brightness,

synchrotron-based, mini-focus EXAFS on anhydrous cryo-preparations to: (i) map with high spatial

resolution the co-distributions of Ca, Cd, Pb, and Zn (as well as P and S) in a linear transect across

the absorptive intestinal epithelia and chloragogenous tissue of hyperbarically quench-frozen

Dendrodrilus rubidus; (ii) determine the ligand-binding speciation of Zn in situ in cryostat sections

of the gut contents and chloragocytes of Lumbricus rubellus quench frozen immediately upon

recovery in the field in order to maintain the fidelity of subcellular metal compartments; (iii) to

recover the non-destructively analysed micro-focus sections for confirmatory morphological and

microprobe assessment in a field-emission scanning electron microscope. We offer three major

conclusions of ecotoxicological and ecophysiological consequence. First, Pb and Zn are almost

exclusively sequestered by oxygen-donating ligands in earthworm tissues, whilst Cd is sequestered

by thiols (i.e S-donating ligands. Second, there is a neglected cell type in the intestinal wall which is

distinct from the predominat ciliated absorptive epithelial cells; it possesses the hallmarks of a cell

playing a significant role in metal sequestration and possibly excretion. Third, the gut contents of the

two epigeic species have different mineral morphologies and compositions. These observations

present a challenge in the age of ―omics‖ technologies to determine the transcriptomic profiles of

functionally distinct cohorts of cells in complex solid tissues.

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5th to 10


Oral – Session 2

Earthworm immunity: a tale of survival by innate but not adaptive

immunity

Cooper Edwin L.

Lab. of Comparative Neuroimmunology, Dept. of Neurobiology, Univ. of California, USA

The immune system of earthworms did not just arise de novo nor were the prescient investigations

during the early 1960s entirely unique since self-nonself recognition as revealed by transplantation,

had been attempted earlier. In Germany about the time of Metchnikoff, there was a prominent school

that devoted considerable effort to transplantation in earthworms. Later in the late 1960‘s

simultaneously in France and in my lab, graft rejection was mediated by cells of the immune system.

Proliferative responses were detected in free cells during xenograft rejection and stimulation of DNA

synthesis was revealed in MLR-like reactions. It is significant that responding and stimulated cells

were characterized as lymphocyte-like coelomocytes (8 to 12 μm). β2-microglobulin-like molecules

occur on coelomocyte membranes of L. terrestris using polyclonal and monoclonal antihuman β2-

microglobulin antibodies. Only one cell type, which constitutes about 15% was labeled. The Thy-1

gene may be closely related in evolutionary history to the ancestral gene for Ig and MHC. Finding a

Thy-1 homolog in invertebrates may establish whether this molecule is more primitive than Ig and

MHC. Thy-1 homolog has been found in squid, tunicates, and in earthworms. Immune systems of

invertebrates and vertebrates are innate, natural, non-specific, non-anticipatory, non-clonal, and

germ line. Only vertebrates possess adaptive immunity: adaptive, induced, specific, anticipatory,

clonal, rearranging genes. Earthworms possess at least two functional leukocytes, small

coelomocytes, and large coelomocytes that mediate lytic reactions against tumor cells in vitro.

Phagocytosis and natural killer cell responses are effected by distinct coelomocytes. The

chlorogogen cell synthesizes and sheds effector lytic molecules. Among them, three have been

identified and sequenced (fetidins, CCF-1, lysenin) and another has been discovered (eiseniapore),

while three other molecules, H (1) H (2) H (3), share agglutinating and lysing functions. Lumbricin I

is the only known earthworm molecule that is antimicrobial but non-lytic. The coelom and its

cellular and humoral derivatives as a source of various cell lineages distinguished for their stem cell

capability to react with ―non-self‖ is clearly an important component of their capacity to the effect of

immune reaction. That earthworms do not develop cancer may be due to their efficient immune

capability since there is evidence of a reasonably long life span.

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5th to 10



Mucor sp. a Pathogen of Pontoscolex corethrurus and Amynthas corticis

earthworms

Alarcón Gutiérrez Enrique1, Hernández Hernández Christian Arturo

1, Torres Jiménez Maria

Guadalupe1, Perroni Ventura Yareni

1, Barois Isabelle

2, Garcia Pérez José Antonio

2

1

Instituto de Investigaciones Forestales, UV, Xalapa, Ver. Mexico

2 Instituto de Ecología A.C., Xalapa, Ver. Mexico

Mucor sp. attacks a wide organism spectrum: Beetles (Coleoptera), mammals like Platypus

(Oberndorf et al., 1993), vegetable species and humans (Pérez et al., 2001). Mucor species have been

tested as an efficient biocontrol agent against Nematoda, both by in vivo inoculation and in vitro

biotest (Ashoub et al., 2009). However, there are no reports of previous studies of pathogenicity of

Mucor using annelids. This study aims to evaluate the Mucor pathogenicity on two tropical species of

earthworms: Pontoscolex corethrurus and Amynthas corticis from the area of Xalapa, Veracruz,

Mexico. The fungus was isolated from dead P. corethrurus bodies infected with Mucor sp. and

developed one week in PDA medium, at room temperature. For the pathogenicity test, fungus was

grown in PDA medium added with cephalosporin antibiotic at 0.5 g/ml in order to guarantee no

bacterial contamination. Earthworms were collected in the INIFOR (Instituto de Investigaciones

Forestales) area (a patch of pasture land in relict forest mountains), placed in plastic containers and

maintained under a greenhouse until the test. The substrate (Ferralitic soil) was collected from the

same area and sterilized 1 h. at 120°C. To recover the spores, 10 ml of dH2O added with 2 drops of

Tween 80 were added to Petri dish with Mucor (Barriga et al,. 2002). Before the inoculation,

earthworms were washed with a solution of Cl at 1 % and then submerged 10 seconds in the spore‘s

solution. The treatments evaluated were: 1) Adult P. corethrurus with fungi. 2) Adult P. corethrurus

without fungi. 3) Adult A. corticis with fungi. 4) Adult A. corticis without fungi. Five replicates by

treatment were prepared. Dead earthworms were counted at 24, 48 and 72 hours. Preliminary results

indicate that the Cl at 1% is lethal, after 72 hours to both earthworm species, reaching values of 95%

and 70% of death for P. corethrurus and A. corticis. However, the treatment that involves Cl at 1%

and Mucor spores inoculation had the highest death range, reaching values of 95% for both species

(dead earthworms were infected). Apparently, P. corethrurus resists more the attack of Mucor than

A. corticis; that may be due to a difference in the protein cell wall of both earthworm species, since it

has been reported that Mucor degrades proteins. Further studies are necessary to evaluate if Mucor is

a factor that mainly attacks earthworms in a healthy state.

55


5th to 10



In Vitro Assessment of Antioxidant Prospects of Earthworm (Lampito

mauritii, Kinberg)

Balamurugan M. 1, Grdisab M.

2, Cooper Edwin L.

3, Parthasarathi K.

1,

Ranganathan L.S. 1

1 Annamalai University, India

2 Rudjer Boskovic Institute,

3 Laboratory of Comparative Neuroimmunology Department of Neurobiology, University of California, Los

Angeles, CA,USA.

Antioxidant activity determined by peroxide value, free radical scavenging activity, antioxidant

capacity and total phenol content in the coelomic fluid, earthworm paste and earthworm extract in

vitro showed that earthworm extract has significantly more antioxidant potency. Results indicate that

earthworm extract can donate electrons to reactive free radicals converting them into more stable,

non-reactive species and terminating the free radical chain reaction. The prevention of the chain

reaction step by scavenging the free radical species seems to be the antioxidant mode of action of the

earthworm extract.

56


5th to 10



Studies on selective therapeutic properties of earthworm Lampito mauritii

(Kinberg)

Balamurugan M. 1, Parthasarathi K.

1, Ranganathan L.S.

1, Cooper Edwin L.

2

1 Annamalai University, India


Angeles; California, USA

Man‘s use of medicinals is as old as humans who are constantly concerned about the state of health.

This has resulted in many concoctions now known as medicines. The branch of science which deals

with medicines obtained from animals and used in the treatment of ailments and diseases is known as

zootherapy. In modern civilization, zootherapy constitutes a significant alternative among many other

known therapies practiced world-wide. Research attention and actions in the areas of ethnobiology

and ethnomedicine have been amplified greatly in the last decade. Since the inception of the

disciplines, scientific research in ethnobiology and ethnomedicine has made important contributions

to understanding traditional subsistence and medical knowledge and practice. In view of the fact that

ancient animals, their parts and their products comprise the inventory of medicinal substances for

curing various ailments, cultures continue the diverse tradition even now. In India, since time

immemorial, great work exists in Zootheraphy and documented in works like Ayurveda, Shidha and

Charaka Samhita. All this knowledge has once again come to our attention, as there has been some

disenchantment with current allopathic medicine, as it has got its own side effect and in fact has no

cure for various diseases. Therefore people are looking for traditional remedies for the treatment of

ailments. But in India this traditional knowledge is fast eroding due to modernization. The studies on

the therapeutic uses of animals and their body parts have been neglected, when compared to plants.

In India, nearly 15–20 percent of the Ayurvedic medicine is based on animal-derived substances.

Earthworms are the soil macro invertebrate oligochaete annelid, playing a major role in improving

and maintaining soil quality and health for sustainable agricultural activities. We intend to discuss

how earthworms have been used in the past by various civilizations and the recent studies in relation

to our observations on the many roles claimed to be the cure for variety of diseases by earthworms in

different forms- live, dried, powder and extracts. Earthworms apart from being used as food for

humans (by Maoris in new Zealand, Japanese, natives in new Guinea, Africa) and animals because of

their high protein content, are eaten or applied to humans to cure illnesses or diseases such as piles,

fever, small pox, jaundice and removal of bladder-stones. Thus, there is great promise in the use of

earthworms as a novel source of the expanding discipline surrounding nutraceuticals.

57


5th to 10



Life cycle and development of Pontoscolex corethrurus (Müller, 1857) in

tropical artificial soil

Cristhy Buch Andressa, Brown George

Embrapa - Florestas / Universidade Federal do Paraná, Brasil

The earthworm Pontoscolex corethrurus is a peregrine species of the Glossoscolecidae family, native

to the Neotropics. The aim of this study was to evaluate the life cycle of this species in tropical

artificial soil (SAT), the substrate used in ecotoxicological tests, and the influence of food availability

and humidity in its growth. The life cycle was evaluated in four treatments: SAT (120 g) without

additional food (SAT0) (n = 24), SAT with 5 g of horse manure (SAT5) (n = 24), SAT with 10 g of

manure (SAT10) (n = 24) and saturated SAT (with 25% greater soil moisture) with 5 g of manure

(SAT5H) (n = 24). Food was provided every 14 days and the containers maintained at room

temperature (19,8 ± 4,1° C). The earthworms used were obtained from 96 cocoons collected near the

Embrapa Florestas (Colombo-PR-Brazil), at different stages of development. These were classified

into 6 color gradients and compared with the time taken until hatching. On average, white transparent

cocoons hatched at 34 days, white opaque cocoons after 33 days, white cocoons with embryo visible

to the naked eye at 30 days; white with rosy stripe at 20 days; light pink at 15 days, and dark pink at

6 days. The intensity of the cocoon color indicated the degree of embryo development. The cocoons

were placed individually in different treatments and development monitored by registering weekly

changes in biometric and morphological characters from hatching to sexual maturation and

oviposition. The newly hatched earthworms weighed 0,052 g and measured 1,9 cm in length. By the

13th week all juveniles in SAT0 had died and reached a maximum of 2,2 cm length and 0,013 g fresh

weight. For the other treatments, all earthworms had already played (?) at the 44th week, showing

length of 5.4 cm and 0.72 g biomass in SAT10, while in SAT5 had 5.1 cm and 0.61 g in SAT5H, 5,1

cm and 0, 59 g. The growth and weight gain in adults of P. corethrurus responded positively to the

offer of food. P. corethrurus was tolerant of excessive moisture in SAT, with eurihigric behavior,

surviving both dehydration and flooding, in contrast to other species. The life cycle of P. corethrurus

was 12 months in SAT. This substrate showed no limitations for the development of the species,

provided that the food was offered.

58


5th to 10



Identification of gene transcripts differentially expressed in the immune

competent cells (coelomocytes) of Eisenia fetida exposed to metallic

pollution

Cocquerelle Claude1, Elsworth Ben2, Brulle Franck1, Morgan John3, Blaxter Mark2,

Vandenbulcke Franck1

1

PRES University Lille Nord de France, France

2 Institute of Evolutionary Biol., School of Biological Sciences, Ashworth Labs., University of Edinburgh, UK

3 Cardiff School of Biosciences, Cardiff University, Wales, UK

Anthropogenic activities can lead to the accumulation in topsoils of Metal Trace Elements (MTE) to

concentrations often exceeding background levels by orders of magnitude. Excessive MTE exposures

can generate degrees of environmental stress that are likely to have deleterious effects (e.g.

disturbance of reproductive and immune functions) on resident organisms. Thus, it is imperative that

plants and animals living in close contact with metal-contaminated soils are studied in order to better

understand their physiological states, the mechanisms of metal sequestration and detoxification, and

the metal-specific modes of toxicity. The emergence of molecular genetic ―omic‖ techniques applied

to ecotoxicology promises to deliver a more fundamental and holistic understanding of the

mechanisms of action of contaminants on receptor organisms. Indeed, gene expression profiles

represent the first level of integration between environmental stressors and the genome, responses

that subsequently escalate to higher levels of biological organisation. The analysis of changes in gene

expression patterns is, therefore, a powerful tool firstly to diagnose the existence of a stress in

individual members of a population, and secondly to analyze in exquisite detail the molecular nodes

of the stress response. In this context, robust and comprehensive DNA sequence information is a

crucial prerequisite. In the present study, Suppression Substractive Hybridization (SSH) was used to

construct four cDNA libraries enriched in up- or down-regulated transcripts in the immune

circulating cells (coelomocytes) of Eisenia fetida (Annelida; Oligochaeta; Lumbricidae) exposed to

metallic pollution. Worms were exposed to artificial soil contaminated by a realistic mixture of MTE

(Cd = 40 mg/kg; Pb = 500 mg/kg; Zn = 700 mg/kg), i.e. metal concentrations found in a strongly

polluted smelter soil. In total, 20,000 SSH clones (5,000 for each of the 4 libraries) were sequenced,

and analysed using the PartiGene analysis suite; they were decorated with functional annotations

(GO, EC and KEGG identifiers). Precisely 5897 unique gene objects were obtained and added to the

initial database; they are particularly interesting since they have been specifically chosen to be

differentially expressed in coelomocytes following metallic stress.

59


5th to 10



Canonical Wnt Signaling Pathway Governs Head Regeneration in

Aeolosoma viride

Chen Cheng-Yi, Chen Jiun-Hong

Institute of Zoology, National Taiwan University, Taiwan

Canonical Wnt signaling pathway has been proven evolutionarily conserving in several animal phyla.

Besides axis establishment during embryogenesis, this pathway also partakes in adult axis

homeostasis and re-establishment during regeneration process in both hydra and planarian models.

However, little is known about whether there is the same mechanism governing regeneration among

annelids that also possess strong regenerative ability. Herein, we introduce a freshwater annelid,

Aeolosoma viride, (capable of re-growing its lost tail or head within 3 or 5 days) as a new model

animal for analyzing regeneration. To assess the impact of canonical Wnt signaling pathway on A.

viride regeneration, downstream proteins GSK-3(?) and α/β(?)-catenin were chosen as the target

molecules. Alsterpaullone (a novel class of small molecule cyclin-dependent kinase (CDK) inhibitors

and LiCl, which can inhibit GSK-3(?) and in turn activate the pathway, thereby significantly

prohibiting A. viride head regeneration. On the other hand, Quercetin, an inhibitor of α/β(?)-catenin,

blocked the pathway but accelerated the speed of A. viride head regeneration. Knocking down of

GSK-3(?) and α/β(?)-catenin by their RNAi affected regeneration processes as their inhibitors.

Furthmore, after treating A. viride with alsterpaullone and LiCl, cellular α/β(?)-catenin was found to

accumulate in the nuclei of A. viride. These results suggest the involvement of Wnt signaling

pathway in A. viride head regeneration, and this is the first report concerning Wnt involved in annelid

regeneration. Although it is not clear whether Wnt pathway affects A. viride tail regeneration, it is

plausible that the pathway governs the anterior-posterior axis in Aeolosoma as for other counterparts.

60


5th to 10



High tolerance to UV-B irradiation in Pontoscolex corethrurus

Chen Po-Chen

National Taiwan University, Taiwan

Generally, earthworms have been known as nocturnal due to lethal impairment from ultraviolet.

Accordingly, UV-B, not UV-A, can induce segmentation, hemorrhage, etc. in some earthworm

species such as Amynthas gracilis, and Metaphire posthuma. Surprisingly, P. corethrurus showed

less sensitivity to UV-B radiation. The lethal dosage of UV-B to P. corethrurus is four times higher

than that A. gracilis. Because P. corethrurus obviously secretes much more mucus on its surface than

A. gracilis or M. posthuma, its mucus was speculated that might serve as UV blocker. In this study,

the secreted mucus from P. corethrurus can effectively absorb UV-B. Furthermore, UV-B can induce

reactive oxygen species (ROS) leading to severe skin damage due to lipid peroxidation. However, the

lipid peroxidation induced by UV-B in epidermis and muscles of P. corethrurus could be quickly

recovered. Therefore, we propose that P. corethrurus can serve as a new animal model to study UV-

resistant and anti-oxidation.

61


5th to 10



Coelomocytes: the mediators of cellular immunity in earthworms

Engelmann Péter1, Cooper Edwin L.

2, Opper Balázs

1, Németh Péter

1

1 Dept. of Immunology and Biotechnology, Faculty of Medicine, University of Pécs, Hungary

2 Lab. of Comparative Neuroimmunology, Dept. of Neurobiology, School of Medicine at UCLA, Univ. of

California, Los Angeles, CA, USA

Earthworm cellular immunity is maintained by different coelomocyte subgroups (hyaline

amoebocytes, granular amoebocytes and chloragocytes) possessing various immune functions. First,

we started to study the expression of conserved molecules (cell surface molecules, enzymes, peptide

hormones) in coelomocytes by various immuno-morphological and immunochemical methods using

library of mammalian-antigen specific monoclonal and polyclonal antibodies. Among those anti-

TNF-alpha and anti-acid phosphatase antibodies proved to characterize coelomocyte subgroups,

moreover anti-TSH antibodies showed distinct staining patterns in earthworm neural elements.

Second, our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and

to provide evidence that the lytic factors originated from coelomocytes. Cell-free coelomic fluid,

supernatants of short-term cultured coelomocytes, and lysates from coelomocytes - derived by

mechanical and detergent extraction - were used in cytotoxicity assays performed on different

standard mammalian tumor cell lines and mouse fibroblasts. Third, because no clusters of

differentiation were described previously, we produced and characterized a library of monoclonal

antibodies specific in differentiation markers (anti-EFCC clones) raised against coelomocyte

(leukocyte) antigens of Eisenia fetida earthworm. Flow cytometry, immunocytochemistry, and

immunoprecipitation analyzed and confirmed the specificity of anti-EFCC clones. Anti-EFCC mAbs

revealed distinct coelomocyte subpopulations and various staining patterns on tissues. Two

functional assays (e.g. phagocytosis and encapsulation) further characterized EFCC clusters revealing

a common coelomocyte marker and three subpopulation-specific markers. The comparative studies

on different evolutionary stages showed strong earthworm specificity. Moreover, we proved that

intracellular calcium plays a major role in the activation of coelomocytes. Immune molecules such as

phytohemaglutinin and bacterial formyl peptide induce calcium influx in coelomocytes studied by

flow cytometry. Finally, all these facts prove that earthworm coelomocytes are a convenient model to

study the evolution of innate immunity in invertebrates.

62


5th to 10



Aporrectodea trapezoides (Dugès, 1828): life cycle in singletons and twins

Fernández Rosa, Novo Marta, Jesús Juan B., Trigo Dolores, Díaz Cosín Dario J.

Universidad Complutense de Madrid, Spain

The parthenogenetic earthworm Aporrectodea trapezoides (Dugès, 1828) is widely distributed all

over the world due to European agricultural practices. In order to provide baseline life cycle data,

cocoons (n= 241) were obtained from field-collected individuals and their features and viability,

incubation period, number of hatchlings and mortality rate were recorded. Singleton and twin

earthworms were cultured from hatching during a 490-day period under controlled conditions with

biomass, survival, reproductive condition and cocoon production being recorded at intervals of 15

days. As a complementary growth measure, instantaneous growth rates (IGRs), assuming that growth

proceeds logistically rather than linearly, were calculated for each 15-day growth interval. The

growth of A. trapezoides followed a sigmoid curve, with a slow initial phase over 60 days, an

exponential phase over 285 days, and a stationary growth phase reached after 345 days. On average,

individuals of isolated-reared A. trapezoides reached maturity at day 153 and body weight at maturity

was approximately 1 g. Growth rates and fecundity of A. trapezoides were not influenced by the

individual status of singletons or twins, although singletons had a mean weight almost twice that of

twins. After the first fifteen days, weight was not significantly different between both types of

individuals, but mortality rates were higher in twins during this initial period. The addition of twins

theoretically would provide an advantageous increase in the population during the colonization of

new areas, when the number of individuals would outweigh the advantage of individual size. In A.

trapezoides however, things seem to be different: twin/singleton status does not have an influence on

individual size, but does influence mortality rate during the first fifteen days of laboratory life. Thus,

a trade-off seems to exist between the number of hatchlings per cocoon and mortality rates during the

first days.

63


5th to 10



Parthenogenesis in Aporrectodea trapezoides (Dùges, 1828): reproductive

traits in field-collected and laboratory-reared earthworms.

Fernández Rosa, Novo Marta, Fernández Marchán Daniel, Simancas Bárbara,

Díaz Cosín Dario J.


Polymorphic degradation of reproductive structures, as often found in other parthenogens, has not yet

been recognized in Aporrectodea trapezoides. Both primary and secondary male sexual characters

such as perithecal papillae, tubercula pubertatis, spermathecae, swollen male porophores and seminal

vesicles are retained in this species. Two possible hypotheses could be the recent appearance of

parthenogenesis or the unusual resistance to mutation in those organs. As reproduction in this species

has been poorly studied, neither pseudogamy nor the existence of stimulating requirements to trigger

its reproduction have never been proved. Experiments on life cycles have usually been done only

with field-collected individuals or with laboratory-reared ones. Hence, some aspects have been

poorly tested, such as the existence of putative learning processes in the field or differences between

potential and real growth or reproduction. In order to record reproductive traits and differences

between field-collected and laboratory-reared individuals of Aporrectodea trapezoides, forty

microcosms with an isolated earthworm (20 with field-collected individuals and 20 with laboratory-

reared ones) and forty containing groups of three (20 with each type of individual) were maintained

during a complete year under controlled conditions. The amount of soil per individual was the same

in both types of microcosm. The number of cocoons was recorded every 15 days by replacing the soil

and wet-sieving the discarded one. Both all individuals kept in isolation and those cultured in groups

produced cocoons, hence completely proving the obligatory parthenogenetic reproduction in this

species without copulation or need of any physical-chemical stimulus. In general, isolated

earthworms produced a significantly higher number of cocoons than those in groups of three, and the

same was recorded for laboratory-reared earthworms when compared with field-collected ones. Not

only is A. trapezoides one of the most abundant earthworm species, but it is also very successful

under different environmental conditions. Thus, new knowledge about reproduction in a possible key

species could be extremely useful in soil management and applied studies.

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5th to 10



Detection of pattern recognition receptors (PRRs) and their associated

signaling pathways in earthworms

Francis Joe W.1, Cooper Edwin L.

2, Wood Todd C.

3



Angeles; California,USA

3 Bryan College, USA

Pattern recognition receptors (PRR) which play a central role in the recognition response of the

innate immune system have been detected in the tissues and immune cells of invertebrates including,

C.elegans, Ciona intestinalis, Chlamys farreri , S. purpuratus, Capitella and Helobdella. The most

commonly detected invertebrate PRRs can be categorized into three major classes: TLR (Toll-like

receptors), NLR (NOD-like receptors) and SRCR (Scavenger receptors). All three receptor classes

bind to pathogen associated molecular patters (PAMPs). A soluble PRR, CCF-1 has been isolated

from earthworm coelomocytes. In addition, several cell surface PRRs including Toll-like receptor 2

(TLR2) and an uncharacterized flagellin-binding protein have been detected using fluorescent

antibody labeling of cell surface components on earthworm coelomocytes. However many antibodies

to mammalian TLRs have not detected TLRs on the coelomocyte cell surface. Sequence analysis of

the recently elucidated L. rubellus genome show that several homologues of TLRs and SRCRs may

exist in L.rubellus. In addition we have been able to detect a variety of intracellular signaling

molecules involved in PRR-dependent immune cell activation, including NFkB, IkB and homologues

of the entire C.elegans based PKM-1 (map kinase) pathway. Some signaling factors associated with

PRRs like MyD88 and others have not been located in the L. rubellus genome. These data suggest

that earthworms may possess both an ancient signaling pathway (PMK-1) and mammalian –like

signaling pathway as part of the PRR-dependent innate immune response.

65


5th to 10



Earthworms, Eisenia and 5 more E’s: A constructivist approach to

teaching invertebrate immunology in an advanced undergraduate

curriculum

Fuller-Espie Sheryl L.

Cabrini College, Radnor, Pennsylvania, USA

The laboratory module describes a flow cytometry exercise appropriate for an advanced

microbiology course designed to familiarize students with innate immune mechanisms and acquire

quantitative reasoning skills. This activity encourages inquiry-based thinking and incorporates an

instructional model based on the 5 E‘s encompassing engagement, exploration, explanation,

elaboration and evaluation. Leukocytes (coelomocytes) isolated from Eisenia hortensis (earthworm)

are used to demonstrate the ingestion of fluorescent Escherichia coli (E. coli/GFP) prey, and the ease

with which this invertebrate model can be adapted for undergraduate teaching of phagocytosis.

Students learn how to set up in vitro cellular assays, are introduced to the basic theory of flow

cytometry, and employ Cell Quest Pro and WinList software programs for the acquisition and

analysis of list mode data files. Single parameter histograms based on fluorescence and dual

parameter dot plots based on light scatter and fluorescence are generated for statistical analyses.

Gating options permit students to restrict their analyses to particular subsets of coelomocytes in

phagocytosis assays. The phagocytosis assay can be modified easily to incorporate hypothesis-driven

predictions providing an extension to this module ideally suited for courses interested in promoting

an inquiry-based learning approach to teaching at the undergraduate level. Assessment of student

learning outcomes is accomplished through a pre-test/post-test evaluation, a student evaluation

questionnaire, and a grading rubric used for evaluation of formal laboratory reports. Data is presented

that illustrates achievement of specific student learning outcomes prescribed for this activity.

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5th to 10



Using dihydrorhodamine 123 to measure reactive oxygen species

production in Eisenia hortensis during phagocytosis: A flow cytometric

method to study innate immunity in invertebrates

Fuller-Espie Sheryl L., Bearoff Frank M.

Cabrini College, Radnor, Pennsylvania, USA.

Phagocytosis is a major mode of immunity for cells (coelomocytes) of the innate immune system of

the earthworm Eisenia hortensis. Phagocytic cells provide non-specific immunity against

environmental hazards and are easily extruded from the dorsal pores for experimental manipulation.

Hyaline amoebocytes, one of three different types of coelomocytes, engulf and destroy bacteria via

phagocytosis with generation of accompanying reactive oxygen species (ROS). Results of several in

vitro experiments will be presented investigating the production of ROS in hyaline amoebocytes

(also known as large coelomocytes) in response to stimulators such as bacteria including Bacillus

megaterium and Pseudomonas stutzeri, as well as the immune suppressing compound corticosterone

(CORT). Large coelomocytes were isolated during analysis based on their size and relative

granularity using flow cytometric methods in which the level of ROS production was measured by

the fluorescent compound dihydrorhodamine 123 (DHR123). Results indicate that ROS production is

best stimulated by introducing bacteria at multiplicities of infection under 100:1 where response rates

were as high as 67%. Preliminary finding show that pretreatment with CORT does appear to suppress

the production of ROS; however, more experimentation is needed to further investigate this

phenomenon.

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Earthworm calcium carbonate granule production does not appear to be

involved in osmoregulation

Hodson Mark E.1, Lambkin Denise

1, Gwilliam Kerry

1, Layton Caroline

2, Canti Matt

3,

Piearce Trevor4

1 Dept. Soil Science, University of Reading, UK

2 Dept. Geography, University of Reading, UK

3 English Heritage, UK

4 Lancaster Environment Centre, University of Lancaster, UK The reason for the secretion of calcium carbonate granules by earthworms is not clear. Previous

studies have suggested a variety of functions for the calcite granules including the regulation of

excess Ca, the regulation of CO2 in tissue fluids and the neutralisation of gut pH (e.g. Piearce 1972, J.

Animal Ecol 41 167–188). None of these proposed functions have been satisfactorily confirmed. A

recent article in Science (Wilson et al. 2009, Science 323 359–362) reported that many marine fish

have an osmoregulatory mechanism that culminates in the excretion of small particles of calcium

carbonate. Since earthworms are essentially aquatic organisms living in the soil we have carried out

experiments to determine whether calcium carbonate granule secretion is part of a similar

osmoregulatory mechanism. Two different experiments were conducted in which earthworms were

exposed to conditions in which osmotic pressure gradients would have existed between the

earthworms and their host soil. Soil from the Hamble soil series (UK) was collected, air dried and

sieved to eat between treatments in either the variable soil moisture experiment (1.9 +/- 0.2 mg

calcite / earthworm / day, n = 17) or in the variable ionic strength experiment (1.5 +/- 0.3 mg calcite /

earthworm / day, n = 18). We conclude that granule production is unlikely to play a significant role in

earthworm osmoregulation.

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Response on the levels of proteins, cells and organisms of Allolobophora

chlorotica exposed dermally to Ni, Cu and Cd ions

Homa Joanna1, Szczesny Ewa

1, Zolkiewicz Aleksandra


2,

Morgan John3, Plytycz Barbara

1

1 Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, Krakow, Poland

2 Pharmaceutical Sciences Reserch Division, King‘s College London School of Biomedical & Health Sciences,

London, UK

3 Cardiff School of Biosciences, Cardiff University, Cardiff Wales, UK

During our previous experiments on earthworms exposed to filter papers soaked with metal chlorides

it turned out that the endogeic species, Allolobophora chlorotica, is more susceptible to metal

toxicity than the epigeic species, Eisenia fetida and Dendrobaena veneta. Therefore the aim of the

present experiments was to study effects of Ni, Cu, and Cd chlorides on A. chlorotica at the levels of

whole organisms, immune-competent cells - coelomocytes, and stress proteins. Adult (clitellate)

individuals of A. chlorotica were collected in the garden of the Institute of Zoology in Krakow.

Worms were kept in the laboratory in their native metal-free soil samples at 16 oC, 12:12 light-

darkness. For experiments, worms were inserted individually into test tubes lined with filter papers

soaked with metal (Ni, Cu, Cd) chlorides, at metal concentrations of 11-176 µg/ml, or with water

(controls). Additional control worms were taken directly from the soil. After 3-day exposure,

earthworm were inspected for viability and anatomical abnormalities. Survivors were used for

coelomocyte retrieval by a mild electric shock (1 min, 4.5V) and/or fixed for morphological studies.

Extruded coelomocytes were counted and used for 1) cytospin preparations; 2) flow cytometric

analysis of percentages of eleocytes, or for measurement of neutral red uptake during in vitro

incubation; 3) preparation of coelomocyte lysates. Immune-blot and immunohistochemistry were

applied to estimate expression of heat shock proteins (HSP72) and metallothioneins isoform 2 (MT-

2) in cell lysates, on coelomocyte cytospins, and on cryostat cross-sections through posterior parts of

worm bodies. The high metal concentrations (especially Ni and Cu) were lethal to worms or induced

drastic anatomical changes, including abnormal swelling, lesions, and/or fragmentations of posterior

segments. Morphological abnormalities were less drastic at lower metal concentrations.

Coelomocytes retrieved from Ni- and Cu-exposed survivors were less numerous and had impaired

ability of neutral red uptake. Cd-exposure affected more neutral red uptakes then the cell numbers.

Expression of HSP72 and MT-2 was evident not only in coelomocytes of survivors, but also on

cross-sections through chloragogen tissue, typhlosole, epithelial cells of intestine, and metanephridia.

In conclusion, whole body homeostasis was significantly disrupted in worms exposed to toxic

chemicals. Acknowledgements: Supported by PB 3502/PO1/2007/32.

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Riboflavin accumulation in chloragogen tissue and chloragocyte-derived

eleocytes in several earthworms species

Homa Joanna1, Mazur Agnieszka Irena

1, Tylko Grzegorz

1, Rosciszewska Elzbieta

1,

Kruk Jerzy2, Klimek Malgorzata

1, Morgan John

3, Plytycz Barbara

1

1 Institute of Zoology; Jagiellonian University, Krakow, Poland

2 Fac. of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland


We have shown previously that riboflavin (vitamin B2) accumulates in chloragosomes of

chloragocyte-derived eleocytes freely floating in coelomic cavity of several - but not all - earthworm

species. Riboflavin plays an important role in immunity, thus its storage in immune-competent cells

may have an adaptive value for earthworms vulnerable to soil-derived pathogen invasion. Thus, it is

important to determine whether riboflavin stores are also present in earthworm species with low

eleocyte counts, especially in chloragocytes. Experiments were performed on adult (clitellate)

earthworms from several species, some of them with an abundance of eleocytes (Eisenia andrei,

Dendrobaena veneta, Allolobophora chlorotica) and others with few eleocytes (Lumbricus terrestris,

L. rubellus, and Aporrectodea caliginosa). Freely floating coelomocytes (consisting of amoebocytes

plus species-specific numbers of eleocytes) were extruded by mild electric shock (4.5V, 1 min). The

worms were then anaesthetized by cooling and the chloragocytes were mechanically detached into

buffer. Both cell suspensions were analyzed by flow cytometry (for cell size, complexity, and

autofluorescence) and spectrofluorometry (for riboflavin content). Transverse cryostat sections

posterior to the clitellum of some worms were prepared for fluorescence microscopy, scanning

confocal microscopy, or transmission electron microscopy. Ultrathin sections revealed that

chloragogen tissue of L. terrestris is more compact than that of E. andrei. Fluorescence microscopic

observations of sections and analyses of cell suspensions revealed distinct riboflavin-derived

autofluorescence in free eleocytes but not in chloragogen tissue of E. andrei. Riboflavin signatures

were also detected in detached chloragocytes, but not in the coelomocytes (i.e. almost exclusively

amoebocytes) expelled from coelomic cavities of L. terrestris, L. rubellus, and A. caliginosa.

Riboflavin was also a prominent constituent of free coelomocytes (i.e. amoebocytes plus eleocytes)

and chloragocytes of D. veneta and A. chlorotica. In conclusion, all investigated earthworm species

have the ability to store riboflavin in chloragocytes and/or chloragocyte-derived eleocytes.

Acknowledgements: This work was supported by a grant (N N303 089834) from the Ministry of

Science and Education (Poland) (for BP and AIM) and by the Jagiellonian University

(K/ZDS/000784).

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RSC96 Schwann cell proliferation and survival induced by earthworm

extract through PI3K/Akt signaling mediated by IGF-I.

Kuo Wei-Wen1, Chang

Yung-Ming

2, Tsai Fuu-Jen

2, Chen

Yueh-Sheng

2, Cooper Edwin L.

3,

Tsai Chang-Hai

4, Huang Chih-Yang

2

1 Department of Biological Science and Technology, Asia University, Taiwan

2 Graduate Institute of Chinese Medical Science, Asia University, Taiwan

3 Lab. of Comparative Neuroimmunology, Dept. of Neurobiology, Univ. of California, USA

4 Department of Health and Nutrition Biotechnology, Asia University, Taiwan

Schwann cell proliferation is critical for the regeneration of injured nerves. Earthworms are widely

used in Chinese herbal medicine to remove stasis and stimulate wound-healing functions. Exactly

how this Chinese herbal medicine promotes tissue survival remains unclear. The aim of the present

study was to investigate the molecular mechanisms by which earthworm extract promotes neuron

regeneration. Our results show that treatment with earthworm extract induces the phosphorylation of

the insulin-like growth factor-I (IGF-I)-mediated phosphatidylinositol 3-kinase/ serine-threonine

kinase (PI3K/Akt) pathway, and activates protein expression of cell nuclear antigen (PCNA) in a

time dependent manner. Cell cycle analysis showed that G1 transits into the S phase in 12 to 16 h,

and S transits into the G2 phase 20 h after exposure to earthworm extract. Strong expression of cyclin

D1, cyclin E, and cyclin A occurs in a time-dependent manner. Small interfering RNA (siRNA)–

mediated knockdown of PI3K significantly reduced P13K protein expression levels, resulting in Bcl2

survival factor reduction and a marked blockage of G1 to S transition in proliferating cells. These

results demonstrate that earthworm extract promotes the proliferation and survival of RSC96 cells via

IGF-I signaling. The mechanism may be primarily dependent on the PI3K protein.

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Cadmium Affects PKC Activity by a Zinc- or Calcium-Independent

Mechanism in Earthworms

Li Bin-Yu

Institute of Zoology, National Taiwan University, Taiwan

Earthworms have been recognized as an important model animal on soil toxicology. Earlier

researches revealed that earthworms have many behavioral and physiological changes in response of

heavy metal such as cadmium. Expression of metallothionein (MT), a small cystein rich divalent

metal ions-binding protein, is among one of those responses induced by cadmium. In vertebrates,

induction of metallothionein caused by protein kinase C (PKC) activator has been demonstrated, but

inductive effects of PKC inhibitor have also been reported. PKC is a very important relay protein of

the signaling pathways which mediate responses such as cell proliferation, apoptosis and smooth

muscle contraction. These responses can be affected by cadmium exposure. However, few

experiments have been done to elucidate how the activity of PKC is affected by cadmium. In this

study, earthworm Eisenia andrei treated with 75ppm cadmium (w/w) for 48 hours significantly

reduced its PKC alpha activity compared to control group. This result was confirmed in vitro by

using 0.04 mM cadmium to treat earthworm tissue crude extract. Furthermore, both calcium and zinc

ions are crucial for PKC‘s activity, and both similar as cadmium ion are divalent cations. However,

PKC alpha activity reduced by cadmium could not be rescued by calcium or zinc ion. This result

suggests that the PKC alpha activity reduction is not caused by competition of cadmium to calcium-

or zinc-binding sites in PKC.

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Schwann cell migration induced by earthworm extract via activation of

PAs and MMP2/9 mediated through ERK1/2 and p38.

Liu Jer-Yuh1, Chang Yung-Ming

2, Tsai

Fuu-Jen

2, Chen Yueh-Sheng

2, Cooper Edwin L.

3,

Tsai Chang-Hai4, Huang Chih-Yang

2

1 Graduate Institute of Cancer Biology, Taiwan

2 Graduate Institute of Chinese Medical Science, Asia University, Taichung, Taiwan


Angeles; California,USA

4 Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

The earthworm, which has stasis removal and wound healing functions, is a widely used Chinese

herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves.

Schwann cells provide an essentially supportive activity for neuron regeneration. However, the

molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we

investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-

degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover,

earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream

signaling expression of PAs and MMPs in a time-dependent manner. Earthworm stimulated ERK1/2

and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in

migration and uPA related signal pathway inhibition. The results were confirmed using small

interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate

Schwann cell migration and upregulate PAs and MMP2/9 expression mediated through the MAPK

pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38) -PAs

(uPA, tPA)- MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms

might play a major role in Schwann cell migration and nerve regeneration.

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The tale of a lazy worm: Reproductive behaviour of Hormogaster elisae

revealed by microsatellite markers

Novo Marta, Fernández Rosa, Jesús Juan B., Almodóvar Ana, Díaz Cosín Dario J.


Endogeic earthworms inhabit the sub-surface layers of the soil, rarely reaching the surface, which

makes their observation and consequently their behavioural study extremely difficult. That is the

reason why in sexual selection studies epigeic and anecic earthworms have been chosen as model

organisms. Nowadays, the development of genetic tools such as microsatellite markers gives the

opportunity to investigate the mating behaviour and sexual selection of these earthworms without the

need for observation. In this study, we address some questions on precopulatory and postcopulatory

sexual selection of Hormogaster elisae, an endogeic earthworm, such as what is the average number

of partners, whether worms choose partners taking into account their size, whether mating success

depends on their size or their genetic variability or whether they use the different spermathecae to

store sperm from different partners. By sampling different points within a population we also tried to

detect the genetic structure of H. elisae at a microgeographical scale. Six to ten mature individuals

were collected from nine different 1m² plots, each one separated by 8 m, within a square of 16 m².

The 75 individuals collected were dissected, and five different samples were taken from each

specimen: a portion of their tegument and their four spermathecae, resulting in a total of 375 samples.

Four microsatellite markers (Novo et al. 2008) were used to analyze all the samples separately. The

results showed that overall genetic diversity values were high, and departure from Hardy-Weinberg

Equilibrium was detected. H. elisae individuals have multiple mates, normally maintaining the sperm

of two individuals mixed in their four spermathecae, with no signs of differential storage of sperm

from different partners. A negative correlation was found between weight differences and mating

success, which seems to indicate that H. elisae individuals select partners with a similar weight.

Their mating success was not dependant on their weight or degree of heterozygosty. The significant

result of a Mantel test indicated isolation by distance, which could mean that H. elisae does not need

long-distance dispersion to seek a partner, and moderate inbreeding was detected although there was

not genetic structuring within the studied area.

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Effects of soil quality on coelomocytes from eco-physiologically contrasting

lumbricid species

Plytycz Barbara1, Podolak Agnieszka

1, Piotrowska Elzbieta

1, Klimek Malgorzata

1, Klimek

Beata Anna2, Szymaski Wojciech

3, Kruk Jerzy

4, Morgan John

5

1 Institute of Zoology, Jagiellonian University, Krakow, Poland

2 Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland

3 Institute of Geography and Spatial Management, Jagiellonian University, Krakow, Poland

4 Fac. of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

5 Cardiff School of Biosciences, Cardiff University, Cardiff, UK

We have shown previously that coelomocytes non-invasively retrieved from earthworm coelomic

cavity may serve as bioindicators of soil pollution: eleocytic riboflavin content in Dendrodrilus

rubidus was high in worms from unpolluted reference soil but low in worms living in, or

experimentally transferred to, either Zn/Pb-contaminated or Ni-contaminatedfield soils. The aim of

the present study was to determine whether coelomocytes in eco-physiologically contrasting

lumbricid species (Eisenia andrei, Dendrobaena veneta, Allolobophora chlorotica) are also affected

by exposure to various metalliferous mine-associated field soils from the UK and Poland, or to metal-

free soils of different physicochemical character. At the end of 3 to 8 week exposures, coleomocytes

were counted and analysed by flow cytometry (for cell size/complexity/autofluorescence), and by

spectrofluorometry (for riboflavin content). Investigated metals accumulated avidly in whole-worm

tissues in species-related manners. Despite metal bioaccumulation, the spectrum of cytotoxic effects

caused by elevated soil metal content on the immuno-competent cells were consistently less severe

than those observed previously in D. rubidus. In particular, the coelomocytes of E. andrei were

slightly affected after 8-week maintenance on Zn/Pb-polluted mine soil from the UK; A. chlorotica

cells were slightly affected after 3-week exposure to the same Zn/Pb-rich soil, but died after short

exposure to aerially contaminated Ni-smelter soil. Ni-smelter soil was not conspicuously harmful to

E. andrei or D. veneta during 3-week exposure. Most revealing were 4-week exposures of the three

species to either Zn/Pb/Cd-contaminated soils from sites in Poland, or to an unpolluted commercial

soil, or to unpolluted field soils of differing geochemistry. D. veneta coelomocytes were unaffected

by maintenance in a variety of soils; in contrast, the riboflavin content was increased (E. andrei) or

decreased (A. chlorotica) in coelomocytes retrieved from worms kept either in metalliferous Polish

(Bukowno) soils, or in unpolluted sandy-clay and loamy-sand soils. In conclusion: (a) effects of soil

quality on earthworm coelomocytes are species-specific; (b) riboflavin content is the most sensitive

of the investigated coelomocyte parameters; (c) riboflavin content is affected not only by soil metal

(Cd, Ni, Pb, Zn) pollution, but putatively also by other ―natural‖ edaphic variables.

Acknowledgements: Supported by PB3502/PO1/32.

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Species-specific sensitivity of earthworm coelomocytes to heavy metal

pollution

Plytycz Barbara1, Klimek Malgorzata

1, Homa Joanna

1, Kruk Jerzy

2, Morgan John

3

1 Institute of Zoology, Jagiellonian University, Krakow, Poland

2 Fac. of Biochem, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland


Earthworm immune-competent cells (coelomocytes) are affected by soil quality, including metal

contamination. Thus it is important to study the effects of individual components of the pollutant

cocktails that are often found in metalliferous field soils. Aporrectodea caliginosa versus

Dendroabena veneta and Allolobophora chlorotica are representatives of two contrasting groups of

earthworms with respect to coelomocyte traits. In A. caliginosa they contain almost exclusively

amoebocytes; both amoebocytes and chloragocyte-derived, riboflavin-storing, eleocytes are well

represented in A. chlorotica and D. veneta. Adult (clitellate) worms of the three investigated species

were exposed for 2 days to filter paper soaked with chlorides of Ni, Cu, Zn, Cd, or Pb at the same

molar concentration (1 mM) (equivalent to 58.7, 63.5, 65.4, 112.4, and 207.2 g/ml, respectively).

Coelomocytes were expelled, counted and analysed ex vivo after electro-stimulation. In A. caliginosa

the total number of amoebocytes was increased in Pb-treated worms; they exhibited enhanced

autofluorescence when retrieved from both Cu- and Pb-treated worms; in vitro neutral red uptake

revealed that pinocytosis was more prevalent (peaking after 15 minutes incubation) in metal-exposed

A. caliginosa than in the controls. In D. veneta, amoebocytes were unaffected by metal exposure; Cu-

exposure caused a minor reduction of eleocyte numbers and of their riboflavin content. In A.

chlorotica, the number of coelomocytes (especially eleocytes) and the riboflavin content of

coelomocyte lysates were significantly lowered in Cu-exposed worms, moderately reduced in

response to Ni, Zn, or Cd exposures, and unaffected by Pb. In conclusion, the amoebocytes of the

three species were relatively resistant to acute dermal exposure to divalent metals. However, eleocyte

numbers and their riboflavin content in the endogeic species A. chlorotica appear to hold some

promise as sensitive biomarkers of metal exposure under laboratory conditions. Acknowledgements:

This work was supported by a grant (No PB3502/PO1/32) from the Ministry of Science and

Education (Poland).

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Isotopic analysis (C, N, S) of setae and soft tissues of Lumbricus terrestris

reared on isotopically distinct diets

Schmidt Olaf1, Jacob Marc

1, Lowe Christopher N.

2

1 University College Dublin, Ireland

2 University of Central Lancashire, UK This project had two objectives: 1) to establish if lumbricid setae are permanent or semi-permanent

tissues that could act as biochemical archives of life histories and 2) to measure the isotopic

fractionation (tissue-diet spacing) for C, N and S in earthworms raised on controlled diets. Newly

hatched Lumbricus terrestris were raised individually for 26 weeks on one of two cattle dung diets

that had distinct C, N and S compositions. Two treatment groups (4-5 individuals each) were fed

exclusively on one of the two diets, and three other treatment groups were initially fed with one diet

and switched to the second diet at 2 g body mass, at maturity or one month after maturity. Setae from

anterior and posterior body segments were collected by digesting body segments (without gut

content) in 10% KOH for 4 h, rinsed repeatedly, transferred into a watch glass with some distilled

water and picked out manually under a light microscope. Soft bulk tissue from segments 1-5 were

also collected and freeze-dried. Diets, setae and soft worm tissues were analysed by EA-IRMS

(Elemental Analysis - Isotope Ratio Mass Spectrometry). C and N isotope results of setae from

worms that had been switched between diets showed that setae are not permanent but turned over at

similar rates to soft tissues. This turnover or replacement could be due to wear and tear, breakage or

growth. This non-permanency implies that lumbricid setae cannot be used as an archival tissue for

biochemical analysis, for example to age-date individuals or detect ontogenetic diet changes.

Measurement of the isotopic composition of soft tissues of the group on the constant control (natural

abundance) diet made it possible, for the first time, to determine isotopic fractionation in earthworms.

The tissue-diet spacing for C, N and S was 2.47, 2.83 and -1.03 per mil, respectively, for the control

diet. These figures will be useful for the interpretation of future studies on the feeding ecology in

earthworms based on natural abundance stable isotope measurements.

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Reproductive seasonality of the Taiwanese montane earthworm Amynthas

hohuanmontis Tsai et al., 2002 (Oligochaeta, Megascolecidae)

Shen Huei-Ping1, Tsai Chu-Fa

2, Chen Jiun-Hong

1

1 Institute of Zoology, National Taiwan University, Taipei, Taiwan

2 Habitat and Ecosystem Division, Endemic Species Research Institute, Jiji, Nantou, Taiwan

Reproductive seasonality of a field population of Amynthas hohuanmontis Tsai et al., 2002, a

Taiwanese montane earthworm belonging to the Pheretima complex of the family Megascolecidae,

was investigated. Its reproductive organs show various stages of degeneration in size, number and

structure. The spermathecae vary from absence (athecal) to three pairs in VI-VIII (sexthecal), and the

prostate glands from large, asymmetrical, to totally absent. For the specimens examined so far, there

were 56.7% athecal, 23.3% sexthecal and 20% between. The number of mature ova of athecal

individuals peaks in summer, whereas that of sexthecal individuals is highest in winter.

Spermatogenesis occurred throughout the year, with highest activity in summer and autumn for

athecal and sexthecal individuals, respectively. Sperms were present in testes/seminal vesicles but no

sperm was found in the histological sections of the spermathecae for all specimens examined, which

is an evidence of parthenogenetic reproduction of this species.

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Different expression of fetidin and lysenin in Eisenia andrei and Eisenia

fetida earthworms

Skanta Frantisek 1, Dvorak Jiri

1, Joskova R.

1, Silerova M.

1, Prochazkova P.

1, Mancikova

V. 1, Elhottová Dana

2, Koubová Anna

2, Pizl Vaclav

2, Bilej Martin

1

1 Institute of Microbiology AS CR, Prague, Czech Republic

2 Biology Center AS CR, Ceske Budejovice, Czech Republic

The earthworms are regarded as a model organism of comparative immunology since 1960s when

transplantation experiments were performed and initiated extensive studies of earthworm immunity.

Moreover, official protocols involving screening of earthworm immunological parameters as

markers/indicators of impaired environmental conditions were accepted. The effect of microbial

environment on innate immunity of model earthworms is thus of particular interest. Eisenia andrei

and Eisenia fetida are two closely related epigeic earthworms. Until recently they have been

considered as subspecies (E. fetida andrei, E. fetida fetida). However, molecular analyses of

cytochrome c oxidase have confirmed their identity as distinct species. As these earthworms live in

completely different environment - E. andrei lives in compost and manure, while E. fetida is found in

litter layer in forests with much lower quantity of microorganisms as well as their heterogeneity - our

work was focused on the differences in their antimicrobial immunity. The coelomic fluid of Eisenia

exhibits numerous biological activities including bactericidal and/or bacteriostatic activities against

soil bacteria. Formerly, two major antimicrobial proteins - fetidin and lysenin - were described in E.

andrei. Both proteins are closely related and share biochemical analogies. Here we show that E.

fetida express neither fetidin nor lysenin, although sequences of both molecules were found in their

genomic DNA. Moreover, the expression is not detectable upon the stimulation by microorganisms

isolated from E. andrei worms or the substrate where they live in. The unique occurrence of fetidin

and lysenin expression in E. andrei could reflect their different living conditions. They could have

developed distinct defense mechanisms to better tolerate a wide range of environmental conditions

and their fluctuations.

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Characterisations of an antibacterial peptide isolated from skin secretions

of the earthworm (Eisenia fetida)

Wang Chong, Sun Zhenjun, Zheng Dongmei, Liu Xuelian

China Agricultural University, China

Earthworm Slime Peptide (ESP) was isolated and purified from the skin secretions of earthworm

(Eisenia fetida) by gel filtrations using Sephadex G-75, and G-50 followed by G-25. The Earthworm

Slime Peptide-1 (ESP-1) was derived from ESP, using C-18 reversed-phase high-performance liquid

chromatography (HPLC). It was found that the ESP was composed of 50 residues of sixteen different

amino acids, among which ten residues were serine. Molecular weight of ESP -1 was 5814 D. The

minimum inhibitory concentrations (MIC) of ESP to Escherichia coli, Pseudomonas aeruginosa and

Staphylococcus aureus was found to be 6.25mg/ml, 6.25mg/ml, and 12.5mg/ml respectively, whereas

the MIC of ESP-1 to the same bacteria was 6.25mg/ml, 12.5mg/ml and 50mg/ml respectively.

Therefore, the antibiotic function of ESP was better than that of ESP-1. It follows that from the skin

secretions of earthworm (Eisenia fetida) antibacterial peptide can be isolated and purified; the

antibiotic function of earthworm's antibiotic peptide was a coordinated function from different

components.

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Function of mucilaginous secretions in the antibacterial immunity system

of Eisenia fetida

Wang Chong, Liu Xuelian, Zheng Dongmei, Sun Zhenjun

China Agricultural University, China

The function of skin secretions in the antibacterial immunity system of the worm Eisenia fetida was

confirmed for the first time in this study. These earthworms were infected in a bacterial bath with

Escherichia coli to induce antibacterial skin secretions of E. fetida. These antibacterial skin

secretions were found to be selective. After induction with E. coli, the bacteriostasis of worm skin

secretions on exposure to E. coli and Staphylococcus aureus increased significantly P secretions. Our

results suggest that mucilaginous secretions are an important defense in the antibacterial immunity

system of E. fetida.

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Detection and preliminary characterization of Toll-like Receptors (TLRs)

in Lumbricus rubellus

Wood Todd C.1, Cooper Edwin L.

2, Francis Joe W.

3

1 Bryan College, USA

2 Lab. of Comparative Neuroimmunology, Dept. of Neurobiology, Univ. of California, USA


Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show

homology with the Drosophila TOLL protein. There is mounting evidence that TLRs are essential

for the development of innate immune reactions to pathogenic microbes in mammals. As with other

immune responses, genome-wide analyses strongly support the view that TLRs or related genes are

ubiquitously conserved in the genomes of non-mammalian organisms. TLRs have been reported from

a variety of aquatic invertebrates, but very little information on TLR distribution in terrestrial

invertebrates is known. Previously, we identified TLR homologues from the leech Helobdella and

polychaete Capitella (Davidson et al. 2008). To identify potential TLR homologues in earthworms,

we searched for homologues of human TLR-2 (AAC34133) in the genome of Lumbricus rubellus

(sequencing in progress) using the BLAST server at LumbriBASE

(http://xyala.cap.ed.ac.uk/Lumbribase/index.shtml). We found 20 contigs (a set of overlapping DNA

segments derived from a single genetic source) with significant similarity to the human TLR-2

(E)[?].

https://mail.mednet.ucla.edu/owa/redir.aspx?C=937bb986d1e447889e075d696bb6e260&URL=http%3a%2f%2fxyala.cap.ed.ac.uk%2fLumbribase%2findex.shtml

82


5th to 10



Cadmium induced abnormal muscle contraction in Eisenia andrei

Wu Ya-Fan, Chen Jiun-Hong

National Taiwan University, Taiwan

Cadmium (Cd), a common heavy metal existing in our environmental soil, is a useful material and

has been widely used in our life such as rechargeable nickel-cadmium battery, oil paints, plastics, etc.

However, bio-accumulation of Cd can cause severe toxicity in many organisms, especially terrestrial

animals. Earthworms have been accepted as a bio-indicator for evaluating soil pollution including

heavy metal contamination. In this study, Eisenia andrei was used to analyze Cd-induced acute toxic

response. After exposure to 200ppm Cd, around 60% of E. andrei began to show abnormal

morphology, including skin wrinkle, partial hyperemia, or abnormal muscle contraction of body wall

in 12 hrs. And most of the Cd-treated earthworms died in 24 hrs. In some serious cases, the

earthworm body wall which might be caused by muscle cell lysis resembling a chain that links the

exposed intestine. The posterior part of the chain-like body could be shed later. Based on the data

from flame atomic absorption spectrometry, the chain-like part of the E. andrei obviously

accumulated higher concentrations of Cd than the normal part of the same earthworm. It is not clear

why the absorbed Cd was accumulated in the posterior part of the body to cause the shedding

behavior in E. andrei. On the other hand, because the symptom of the abnormal muscle lysis in E.

andrei caused by Cd shows some similarity as rhabdomyolysis in human, it will be interesting to

study whether the Cd-induced abnormality in E. andrei could serve as an animal mode to study

rhabdomyolysis, the breakdown of fibers and consequent release of its contents into the bloodstream,

which if untreated can result in kidney damage.

83


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Session 3

Earthworms as Indicators of Change on

a Local, Regional and Global Scale

84


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Are earthworms good bioindicators of human activities?

Paoletti Maurizio G.

Padova University

Vincenzo Tanara in 1644 and Charles Darwin 1881 were among the first modern supporters of the

earthworm activity, function and indication in times in which such invertebrates were not much

popular in crop-fields and appreciated only as baites for line fishing and or for chickens.Today

earthworms have increased their rank among most people perception as beneficial for soil, garden

and as fertility promoters. Luckily they have been chosen among 54 potential bioindicators as one of

the key parameters to assess organic versus conventional farming in one European founded project

Bio- Bio. However their snakelike shape and their place of thriving- in the soil make them

―suspicious‖ and ―dirty‖ creatures. For these reasons most earthworm popularization is made with

head and legs making the worms imagine more like vertebrates or humanized pets.These

perceptions are important when considering or promoting earthworms as bioindicators among

farming community or citizens-consumers. The current knowledge of these creatures being very

limited among the large majority of people no matter about the different taxa and or different

behaviours. I will discuss some points of earthworms bioindication in rural and seminatural

landscapes. Different ecological roles and earthworms importance as quality indicators in different

landscapes. Organic Farming activity and earthworms. Earthworms local knowledge and their use

as human food (the cases of New Zealand and The Alto Orinoco). Bioindication limits will be also

discussed for earthworms. The cases in which some anecic species are referred as potential invaders

or pests or are targeted as potential new colonizers to b introduced for tropical countries ―in which

apparently‖ no anecic native species are active.

85


5th to 10


Oral – Session 3

The use of earthworms as tool for soil monitoring, characterization and

risk assessment. Example of a Bioindicator Programme developed at

National scale (France)

Pérès Guénola

1, Vandenbulcke Franck

2, Guernion Muriel

1, Piron Denis, Rougé Laurence

1,

Hedde M.3, Bispo A.

4, Galsomies L.

5, Grand C.

4, Cluzeau Daniel

1

1University Rennes, France

2University Lille Nord, France

3INRA Versailles-Grignon, France

4ADEME, France

5ADEME DABEE-DSQA, France

Soil can be considered as a non-renewable resource that must be protected because it is essential to

our livelihood (it provides food, drinking water, biomass and raw materials) but its degradation is

still accelerating (e.g., erosion, contamination, compaction). Thus, protecting and managing soils

require a set of indicators able to judge the degradation and/or remediation of its properties and

functions. Considering the lack of knowledge on the best bioindicators to describe soil quality, a

national research programme has been set up to develop such indicators (2006-20012). This

―Bioindicator Programme‖ is the first in Europe. The main objectives are to provide new tools for

soil monitoring, characterization and risk assessment, based on soil biological properties. In the first

step of this programme (2006-2009), research teams developed and tested their indicators (n=80) in a

few given situations. After selection, the most relevant indicators (n=22), including earthworms, are

presently being tested and compared in a large number of common contexts involving: 13 sites all

over France (forest, agricultural and contaminated sites) and 51 contexts differing in terms of land

use, contamination origin and pollution level. Earthworms are being studied at community level

(abundance, biomass, species and functional structures) as well as organism level (applying

metallothionein coding gene expression in earthworms). Moreover, the relationships between

earthworms, assessed at functional level (epigeic, endogeic, anecic), and other soil fauna

(collembola, mites, nematodes) are being analysed using multi-parameter analysis. The results

presented in this communication are those obtained during the spring of 2009. They clearly show the

value of the earthworm community as indicator of land use, especially of the impact of different

organic matter inputs, as well as tillage vs. no tillage systems. Moreover, earthworm communities

indicate strongly the level of different sources of pollution (metal or HAP). Furthermore, the first

results obtained on L.terrestrisare encouraging as they show that the expression of metallothionein

increases in contaminated soil. The multi-parameter analysis (HAC) revealed 4 groups which defined

specific indicators (indicator of sites, land uses, pollution). These first results are very interesting

because they reinforce the interest of using earthworms in soil monitoring, characterization and risk

assessment.

86


5th to 10


Oral – Session 3

A conceptual model to understand the role of earthworms as bioindicators

in the Argentine Pampa steppe

Momo Fernando

1, Sandler Rosana

2, Falco Liliana

2

1Instituto de Ciencias - Universidad Nacional de General Sarmiento, Argentina

2INEDES - Universidad Nacional de Luján, Argentina

We present a conceptual model for the response of different earthworm species to soil conditions.

The model considers two main axes: the first represents the soil organic matter content (OM),

associated to the fertility and agricultural aptitude of the soil; the second axis represents the intensity

of disturbance (DI) due to tillage and other agricultural practices. We used information obtained over

the last 25 years from the Pampa region of Argentina under different land uses, from agriculture to

forestry. In this region there are both indigenous and introduced earthworm species. The chosen axes

allow us to define four soil groups (one per quadrant). 1) Lightly disturbed and fertile, located in the

fourth quadrant (high OM – low DI); these are soils under grasslands used for cattle production or

forested soils. 2) Fertile agricultural soils, located in the first quadrant (high OM, high DI). 3)

Explored and poor soils in the second quadrant (low OM, high DI). 4) Poor and undisturbed soils in

the third quadrant (low OM, low DI). Starting with this framework and knowing the ecophysiological

responses of the more frequent earthworm genera, we defined the following characteristic groups:

genera Amynthas and Microscolex for type [1] soils; genus Aporrectodea for type [2] soils; genus

Eukerria for type [3] soils, and genera Octolasion and Bimastos for type [4] soils. Soils having low

OM values contained mainly endogeic and oligohumic species; soils with high OM values showed

poly- or meso-humic species. On the other hand, soils with high DI had earthworms with small body

size and low variance; soils with low DI had large earthworms with high variability in body size.

This conceptual model is a first approximation in an effort to use earthworms as bio-indicators of the

soil history and ecological status. Moreover, the model helps explain field results. This kind of

framework can be represented by a soil mandala using the similar concept of Margalef to explain the

presence of algae groups in marine and freshwater ecosystems.

87


5th to 10


Oral – Session 3

Quality in semi-deciduous forest areas

(Centre-West, Ivory Coast)

Tondoh Jerome E.

1, Guéi Arnauth M.

2, Csuzdi Csaba

3

1CIAT-TSBF, Mali

2UFR des Sciences de la Nature, Côte d'Ivoire

3HAS and Hungarian Natural History Museum, Hungary

Although soil quality has been used as an indicator of sustainable land management for several

decades, there is no common agreement about its evaluation and monitoring. We used here a

multifunctional indicator of soil quality that combined a set of 4 sub indicators associated with

different soil ecosystem services to assess the variation of soil health along a gradient of forest

disturbance in Centre-West Ivory Coast. The sampling method consisted in digging out 3 monoliths

(50 x 50 x 20 cm) at 5 m intervals, collecting soil samples and measuring chemical parameters, bulk

density and resistance to penetration at each sampling point along a gradient of forest disturbance.

The land-use types comprised semi-deciduous forests (as a baseline), tree plantations, fallow,

perennial and annual crops. The calculation procedure was based on a sequence of Principal

Component Analysis (PCA) by which physical, chemical, soil organic matter content and earthworm

diversity sub indicators were combined into a single general indicator of soil quality (GISQ). In

parallel, earthworms were used to test the concept of bio-indicators of soil quality and we sought to

test the theory of species indicators as well. Results showed a significant variation of GISQ along the

gradient of forest disturbance, with the highest values (0.78±0.05-0.93±0.03) obtained in natural

forests, the lowest (0.24±0.05) in recurrent fallows and mixed-crop fields, while tree-based systems

showed intermediary values (0.40±0.03-0.58±0.06). There were significant relationships between

earthworm density (R2=0.27, F=17.2, P=0.0005), biomass (R2=0.35, F=20.2, P=0.0000) and the

GISQ, showing that earthworm communities can be used as indicators of soil quality. Moreover, we

found 6 indicator species among which 4 (Dichogaster terrae-nigrae, Chuniodrilus zielae,

Dichogaster baeri and Millsonia lamtoiana) were associated with tree-based systems and 2

(Millsonia omodeoi and Hyperiodrilus africanus) with recurrent fallows and mixed food crops. The

present study thus revealed that a GISQ can be used to assess forest disturbance and confirmed that

earthworm communities can be used as indicators of soil quality.

88


5th to 10


Oral – Session 3

Earthworm communities in deciduous forest and productive systems in

Yucatan, Mexico. Changes, disturbance responses and indicator species

Bautista Francisco

1, Díaz Castelazo Cecilia

2, García Robles Marisol

3

1UNAM, Mexico

2INECOL, A.C., Mexico

3FMVZ, Universidad Autónoma de Yucatán, Mexico

In Yucatan, Mexico, we studied earthworm communities (biomass, abundance, species composition

and dominance) in deciduous tropical forest and four livestock production systems established

through slash and burn practices, with increasing management disturbance: silvopastoral systems -

SP-, 2 year-old and 12 year-old Taiwan grasslands -TP2 and TP12-, and Star-grass pastures –SG12.

The earthworms were collected in 10 monolith-sampling points per system, in order to detect

sensitive species with potential as indicators of karstic soil health. We also performed discriminant

analysis to assess the biological validation of management practices according to earthworm

communities, and provide a disturbance response index for both native and exotic species. We found

seven endogeic species: Balanteodrilus pearsei, Dichogaster affinis, Dichogaster bolaui,

Dichogaster sp. (Acanthodrilidae), Diplotrema murchiei, Pontoscolex corethrurus

(Glossoscolecidae) and a newly recorded Ocnerodrilidae species. The last four species are new

records for the state of Yucatan. B. pearsei, D. murchiei and the new Ocnerodrilidae species were

native; the remaining species were exotic. B. pearsei was the only species found in every production

system and was the most abundant, dominant species, accounting for 87.5% of the total earthworm

biomass. TP2 had the highest biomass of Oligochaeta, and disturbance response indexes confirmed

that the earthworm community responded positively to intermediate levels of management

disturbance (TP2). A numerical classification of the production systems showed that 62% of the

earthworm species recorded were correctly assigned to production systems and explained 42.2% of

total variance in the classification (Wilk´s lambda = 0.117, p<0.05).

89


5th to 10


Oral – Session 3

Change trajectories of earthworm communities after conversion of mixed

semi-natural broadleaved forest to spruce monoculture along a soil trophic

gradient

Muys Bart

1, Verstraete Gorik

2, Hlava Jakub

3, Valckx Jan

1, Verheyen Kris

2

1Katholieke Universiteit Leuven, Belgium

2Universiteit Gent, Belgium

3Czech University of Life Sciences, Czech Republic

The effect of tree species choice and tree species conversion on soil acidity, humus type and

earthworm communities through the effect of their decomposing litter has been thoroughly studied.

However, the size of such change trajectory as a function of initial soil trophicity has not been looked

at in a systematic way, nor is it well understood whether earthworms play an active role in this

environmental change. The objective of this study was to better understand the change trajectories of

topsoil, humus type and earthworm communities by multiple pair comparisons along a soil trophic

gradient. In a context of acid deposition sensitivity mapping it is common understanding that poorly

buffered soils are more sensitive to acidification, so we hypothesized longer trajectories in poorer

soils. Along a trophic gradient in a large (about 20,000 ha) temperate semi-natural forest in Southern

Belgium we selected 60 plots, half of which in patches of 1-5 ha of Picea abies (Norway spruce) of

approximately 50 year of age, and 50 plots at short distance from each conifer plot in the broadleaved

matrix (main species Carpinus betulus, Quercus robur, Fagus sylvatica, Quercus petraea and

Fraxinus excelsior). In all plots topsoil and substrate chemistry were described, humus form

determined and earthworm communities characterized. Causal relations between factors were tested

with path analysis using structural equation modeling. In general, spruce monocultures acidified

soils, increased ectorganic horizons, decreased earthworm biomass and eliminated endogeic

earthworm species. Interestingly, the change trajectories were larger in richer soils with higher buffer

capacity. This can be interpreted as a regime shift between soil buffer ranges, which is supported by

path analysis suggesting a forward-feeding mechanism of increasing litter accumulation through the

production of organic acids toxic for burrowing earthworm species (endogeics and anecics).

90


5th to 10


Oral – Session 3

Earthworm and other soil invertebrate communities along an altitudinal

gradient in the Silesian Beskid mountains (Southern Poland)

Rozen Anna

1, Mysłajek Robert W.

2, Sobczyk Lukasz,

1Institute of Environmental Sciences, Jagiellonian University, Poland

2Association for Nature WOLF, Poland

The Silesian Beskid range is the western-most part of the Carpathian Mountains. The highest peak in

the range reaches 1,257 m a.s.l. The climate is characterized by high precipitation (1,200 mm per

year) and long-lasting snow cover. The Carpathians are densely forested with a clear altitudinal

zonation of vegetation including: uplands (200-550 m a.s.l.), lower forest zone (dominating beech-

spruce, 550-1,100 m a.s.l.) and higher forest zone (dominating spruce, 1100-1,350 m a.s.l.). The

study on earthworms was carried out along an altitudinal gradient (500-1000 m a. s.l) in three

vegetation zones – both in the meadows and in the forested sites (beech, spruce, oak-hornbeam,

alder-ash). In spring, summer and autumn of 2004, 2005 and 2006 soil animals were studied in 12

plots. On every sampling occasion and in every plot 5 samples (20×20×20 cm) were taken.

Earthworms and other soil invertebrates were separated from soil by hand sorting and preserved. In

the laboratory, earthworms were identified to species level and the other invertebrates to higher

taxonomic levels. The biomass of earthworms and other invertebrates was estimated. Eight

earthworm species were found: Aporrectodea caliginosa, A. rosea, Dendrobaena octaedra,

Dendrodrilus rubidus, Eisenia lucens, Lumbricus castaneus, L. rubellus and Octolasion lacteum. The

highest earthworm density, diversity (H‘) and biomass was in foothill meadows. The earthworm and

other invertebrate community structure was compared between sites and sampling seasons. The

impact of site, season and altitudinal gradient on soil animals communities was analyzed.

91


5th to 10


Oral – Session 3

Using earthworm behaviour (avoidance, cast production and burrowing

activities) to assess the effects of liming on the functioning of

anthropogenically acidified forest soils

Auclerc Apolline

1, Huguier Pierre

1, Capowiez Yvan

2, Guérold François

1, Nahmani Johanne

1

1LIEBE, CNRS UMR, Université Paul Verlaine, France

2INRA, UR 1115 "Plantes et Systèmes Horticoles", France

Liming of acidified ecosystems can represent a valuable alternative to promote the recovery of

biodiversity impoverished by anthropogenic acidification. For the first time in France, two forested

catchments (2x120 ha) located in the Vosges Mountains (NE France) were limed (2.5 t ha-1) by

helicopter in the autumn of 2003. A preliminary study demonstrated higher earthworm abundance in

the limed soils than in the surrounding acid soils, five years after the liming operation. In order to

determine the detailed effects of liming, we carried out three laboratory tests to follow the possible

modifications in the behaviour of three ecological groups of earthworms. Three modalities were

tested: (i) acid soil (pH = 3.9), (ii) in situ limed soils (pH = 4.2) (both of them sampled in the

catchments) and (iii) in vitro limed soils composed by acid soil mixed with liming concentrations (1,

2.5, 10 and 20 t ha-1). First, avoidance tests were performed using Eisenia fetida and Lumbricus

castaneus sampled on the in situ limed site. Ten earthworms were introduced for 24h in plastic

boxes, half composed of acid soil and half by each limed soil. Secondly, L. terrestris were introduced

in Petri dishes to compare the earthworm body mass change and cast production after an exposure for

7 days in these three soils. Finally, Aporrectodea caliginosa, A. giardi or L. castaneus were incubated

for 9 weeks in soil cores filled with acid, in situ or in vitro limed soils and X-ray tomography was

used to assess their burrowing activities. A significant repellence was observed for the acid soil while

the in vitro and in situ limed soils were attractive: 94 ± 5% of E. fetida and 48 ± 27% of L. castaneus

were found in the in situ limed soil. Similarly, 100% of E. fetida and 80 ± 10% of L. castaneus were

found in the in vitro limed soil at 2.5 t ha-1 (pH = 6.2). Cast production of L. terrestris significantly

increased with soil pH when a rational concentration of lime (2.5 t ha-1) was mixed with acid soil

(pH 5.5). Finally, earthworms significantly increased their burrowing activities in the surface layers

of soil cores in the in vitro soil at 2.5 t ha-1 (pH = 5.4). Thus, these studies demonstrated that

earthworm behaviour is a relevant indicator of liming restoration. A study under field conditions with

a local liming operation on an acid catchment is in process to validate these laboratory results.

92


5th to 10


Oral – Session 3

Resilience to inundation in earthworm communities in river floodplain

grassland: trait attributes and diversity

Faber Jack, Lange Marieke de, Kramer Koen

Alterra, Wageningen UR

The relationship between biological diversity and ecosystem functioning can be studied in terms of

ecological traits in species. We studied ecosystem functioning in terms of resilience to stressors,

using as an example an earthworm community in a frequently flooded floodplain in the Netherlands.

Our general hypothesis was that ecological resilience is determined by the distribution of traits

between species within the community: a greater diversity of traits to overcome the adverse effects of

flooding will allow the earthworm community to better respond to inundation and therefore be more

resilient. Whilst earthworms can occur in high densities in floodplains, periodic occasions of flooding

restrict population growth and species diversity. The different mechanisms employed by earthworm

species for coping with this stressor can be described by a combination of ecological traits. For 10

earthworm species encountered along an inundation gradient in the Duursche Waarden floodplain

area flanking the river IJssel we compiled a dataset of 11 relevant ecological traits. Trait frequency

analysis confirmed the presence of flood tolerance traits at more frequently inundated plots, such as

active dispersal, hydrophily, and parthenogenetic reproduction. The greater diversity of flood

tolerance trait attributes in low-elevation plots suggests that earthworm communities at these sites are

more resilient to flooding. Ordination analysis using trait composition instead of species composition

somewhat improved the proportion of variance explained, suggesting that trait composition yields

more information on community resilience than species composition. The combination of the two

approaches improved our understanding of community resilience.

93


5th to 10


Oral – Session 3

Earthworm biological traits, soil and landscape characteristics

Marichal Raphael

1, Praxedes Catarina

2, Brown George

3, Castaneda Edana

4, Desjardins

Thierry5, Gond Valery

6, Grimaldi Michel

5, Guevara Edward

7, Oswald Johan

8,

Sarrazin Max9, Lavelle Patrick

1

1UPMC, France

2Museu Paraense Emílio Goeldi, Brazil

3Embrapa Florestas, Brazil

4Universidad Amazonia, Colombia

5IRD, France

6CIRAD, France

7CIAT, Colombia

8University of Rennes, France

9Centre IRD, Guyana

This study analyzes the links between the occurrence and frequency of biological traits in earthworm

communities and soil and landscape characteristics in Amazonia. Biological traits are a powerful

attribute to consider in order to understand the links between biodiversity and ecosystem processes.

The present consensus is that diversity expressed in the nature and range of traits, rather than species

numbers, determines ecosystem functioning. Biological traits are also important characters that

determine communities in a given environment. Few studies have ever considered earthworm

biological traits and their ecological significance. Therefore, we sampled earthworms at 135 points

distributed among 3 localities, 9 sub localities and 27 farms in the South of Pará state (Eastern

Brazilian Amazonia). At each point, 3 samples 25 x 25 cm in size were hand-sorted for earthworms

(TSBF method). Traits (10) of 1256 earthworms in total were measured. They were individual

(length, post and pre-clitellar diameter, length and type of caudal setae, pigmentation) or species

specific (gizzards, thickness of septa, type of typhlosole, ecological category), multiqualitative, semi

quantitative or fully quantitative. All the earthworms were determined at the morpho-species level.

The landscape in a 100 m radius centered on each point was described with a set of metrics that

basically described the composition, diversity and fragmentation pattern of the landscape on a matrix

of 20 x 20m pixels. At each point 41 chemical and physical soil parameters were measured. Each

data set (trait frequency matrix, landscape metrics and soil data) was analyzed using PCA and co-

inertia analyses were performed to search for co variations. Significant co-inertias were measured

between the biological traits matrix and landscape structure (p=0.047, RV=0.05), landscape

composition (P=0.015, RV=0.12) and soil characteristics (p=0.01, RV=0.26), respectively. These

results show a strong link of earthworm traits with soil characteristics and landscape structure and

composition.

94


5th to 10


Oral – Session 3

An integrated assessment of metal adaptation in the sentinel earthworm

Lumbricus rubellus

Bruford Michael W.

1, Juma Gabriela

1, Donnelly Robert

2, Bundy Jake

3, Kille Peter

1, Harper

Georgina2, Morgan John

1

1Cardiff University, UK

2The University of Glamorgan, UK

3Imperial College London, UK

A central premise of the use of sentinel organisms such as earthworms in toxicity testing is that their

presence or abundance reflects the level of contaminant to which they are sensitive and predicts that

individuals of a given sentinel species are less likely to survive at a contaminated site due to the

physiological consequences of contamination. However, it is well known from pest management and

evolutionary ecology research that organisms are capable of rapid adaptation to toxins via a variety

of mechanisms, enabling them to survive and reproduce in environments where levels of toxicity

would eradicate native populations. Such adaptation has been shown to be a mix of phenotypic

plasticity and adaptive evolution through molecular processes such as gene amplification. If adaptive

evolution is a common feature of sentinel species, it potentially confounds their use in toxicity testing

and makes generalizations among sites and populations problematic. We examined this question in

the earthworm Lumbricus rubellus, using a population adjacent to a long-established Nickel smelter

in Clydach, Wales and two control populations as a model system. We tested the alternative

hypotheses that populations closest to the Ni source were maintained by immigration from less

contaminated sites versus the hypothesis that this population had become adapted to Ni

contamination by rapid evolution. To examine this question we measured gene-flow, genetic

diversity, soil chemistry and metabolite differences between populations of differing distance to the

source and compared these results with 'natural' variation due to soil chemistry and agriculture at two

uncontaminated sites. I will synthesize the results of this study examining the correlation between

patterns of diversity and differentiation using DNA-based molecular markers, the chemical

environment and the metabolome revealed through multivariate NMR spectrum analysis. Preliminary

results indicate that soil chemistry has a strong effect on metabolome profiles but that the classical

patterns of evolutionary adaptation (genetic erosion and limited gene-flow between populations close

to the Ni source) are not evident and that populations in contaminated sites are more likely to be

sustained by immigration. Further analyses are ongoing and will be presented.

95


5th to 10



Population behavior of the order Oligochaeta in a tropical soil under

different corn production systems

Alarcón Gutiérrez Enrique

1, Torres Jimenez Maria Guadalupe

1, Landa Guerrero Yeudiel

1,

Garcia Pérez José Antonio2, Ortiz Ceballos Angel I.

3, De los Santos Bailon Martin

2,

Barois Isabelle2

1Instituto de Investigaciones Forestales Universidad Veracruzana

2Instituto de Ecología, A.C.

3Instituto de Biotecnología y Ecología Aplicada Universidad Veracruzana

Understanding the ecology of soil is crucial for its sustainable management, however, given its

complexity, little is known about the dynamics of the variables under different land uses (Anderson

1975). Agriculture is undoubtedly one of the activities that require attention in the field of species

conservation, since large land areas have been transformed to become growing areas. The

international project "Conservation and Sustainable Management of Below Ground Biodiversity"

propose solutions to conserve species of organisms which live below ground and maintain the

ecological functions of soil. This study, located within the former context, aimed to assess the

population density of Oligochaeta in an experimental plot located in the biosphere reserve "Los

Tuxtlas" in the state of Veracruz, Mexico, under different systems of maize production. In order to

evaluate the relative abundance of earthworms, different treatments and control were used as follow:

Maize-Mucuna (Mucuna pruriens) -Fertilizer (MMF); Maize-Mucuna (GM); Maize-Fertilizer (MF);

Maize (M); Control (C) and uncultivated land (S). The experiment was conducted during a

production cycle of maize in the summer of 2008. A higher density of earthworms was observed in

the uncultivated land, far exceeding all other treatments, reaching a value of 10.34% in relative

abundance of earthworms with respect to the other macrofauna accounted; the treatment that

followed, the Maize without fertilizer, had a value of 3.0%; and finally, Maize-Mucuna with 1.33%.

Results suggest that treatments without management seem to be preferred by the earthworms,

showing a tendency to decrease their populations in the presence of agrochemicals.

96


5th to 10



Earthworm density and biomass in various land use systems in Northern

Paraná State, Brazil**

Bartz Marie Luise Carolina

1, Pasini Amarildo

1, Brown George

2

1Universidade Estadual de Londrina, Brazil

2Empresa Brasileira de Pesquisa Agropecuária, Brazil

Soil alterations, such as oscillations in temperature, moisture, and organic matter contents, mainly

resulting from agricultural activities, can impact earthworm abundance. The objective of this paper

was to evaluate earthworm density and biomass in various land use systems in Northern Paraná State,

Brazil. The study was conducted in Rolândia and Arapongas municipalities, in five areas: 1) no-till

(36 yr) – NT; 2) subsoiled no-till – SNT (every 3 yr); 3) pasture – P (> 30 yr); 4) coffee plantation –

C (> 30 yr); and 5) native forest – NF. Soils of the area were Oxisols: Rhodic Hapludox in NT, SNT,

C and NF and a transition between Rhodic Hapludox and Rhodic Kandiudox in P. Nine samples were

taken in each area over a one-year period (march 2008 to march 2009) every three months, using the

TSBF (Tropical Soil Biology and Fertility) methodology (hand sorting of 25 × 25 cm monoliths from

the 0-20 cm layer of soil). The earthworms were removed from the soil and preserved in 4%

formaldehyde, and then counted (ind. m-2

) and weighed (g m-2

). The mean density in the year was

higher in P (105 ind m-2

), followed by NF, NT and SNT with 15, 13 and 12 ind. m-2

, respectively.

Lowest mean density (4 ind. m-2

) was found in C. Mean biomass followed the same order:

P>NF>NT>SNT>C, with 7.15, 1.07, 0.21, 0.09 and 0.03 g m-2

in each area, respectively. In the five

samples dates (mar/08, jun/08, set/08, dec/08 and mar/09) highest densities and biomasses were

observed in sep/08, in the end of the dry season, in P, C, SNT and NF. In NT, highest values for

density and biomass were observed in mar/08, and then decreasing over the sampling period. All

areas showed a large decrease in density and biomass in dec/08 and mar/09 which was attributed to

climatic conditions during the study period which affected both, the water balance and soil moisture,

determining factors for the survival and reproduction of earthworms. **Financed by Capes

(scholarship for the first author) and Fundação Agrisus (divulgation of the results)

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Earthworm species in various land use systems in Northern Paraná State,

Brazil


1, Pasini Amarildo

1, Brown George

2


2Empresa Brasileira de Pesquisa Agropecuária, Brazil

Earthworms are present in almost all terrestrial ecosystems, and are one of the most important

components of the soil invertebrate macrofauna. Species identification is essential in biological,

ecological, and behavioural studies, as well as to characterize the benefits of these organisms in

various land use systems (LUS). This study aimed to identify earthworm species in four LUS in

Northern Paraná State, Brazil. The study was conducted in the counties of Rolândia and Arapongas,

in five areas: 1) no-till (36 yr) – NT; 2) subsoiled no-till – SNT (every 3 yr); 3) pasture – P (> 30 yr);

4) coffee plantation – C (> 30 yr); and 5) native forest – NF. Soils of the area were Oxisols: Rhodic

Hapludox in NT, SNT, C and NF and a transition between Rhodic Hapludox and Rhodic Kandiudox

in P. Nine samples were taken in each area over a one-year period (march 2008 to march 2009) every

three months, using the TSBF (Tropical Soil Biology and Fertility) methodology (handsorting of 25

× 25 cm monoliths from the 0-20 cm layer of soil). The earthworms were preserved in 4%

formaldehyde, and then they were separated in juveniles and adults and were identified at the genus

or species level when possible. In the five samples dates (mar/08, jun/08, sep/08, dec/08 and mar/09)

highest diversity was founded in P (7 species): Fimoscolex sp. and Glossocolex sp. (native and new

species), Pontoscolex corethrurus, Dichogaster gracilis, D. affinis, D. saliens (exotics) and NIsp.1

(not identified species – native?). In NT, 6 species were founded: P. corethrurus, D. affinis, D.

saliens (exotics), Fimoscolex sp. and Belladrilus (Santomesia) emilianii and Belladrilus sp.1

(natives). In NF, 5 species were found: P. corethrurus, D. affinis (exotics), Urobenus sp., Belladrilus

sp1 and sp2 (natives). Lowest diversity was found in SNT (3 species): D. saliens (exotic), B. (S.)

emilianii and Belladrilus sp1 (native), and in C (2 species): D. saliens (exotic) and B. (S.) emilianii

(native). Therefore, several native species were able to survive in the agro ecosystems, though

disturbance was high, although these tended to be of small size and higher diversity was associated

with lower disturbance, allowing comparable diversity with the native forest. **Financed by Capes

(scholarship for the first author) and Fundação Agrisus (divulgation of the results)

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Earthworm abundance and diversity in no-till systems in SW Paraná

State, Brazil*


1, Pasini Amarildo

1, Brown George

2, Mello Ivo

3, Botoluzzi

Jeankleber3, Tamiozzo Tiago

3, Pereira Pilecco Osivan

4, Toledo Ramiro

4



3Federação Brasileira de Plantio Direto na Palha, Brazil

4Vetagro Consultoria Agronomica, Brazil

No-tillage is a widely practiced conservation agriculture system in Brazil, with over 22 million ha

currently in no-till. This management system, which includes permanent soil cover (straw), minimum

soil physical disturbance and crop rotation with green manures, linked with integrated pest

management, is known to promote, among other factors, the role of soil organisms in soil fertility.

Therefore, the present study was undertaken to assess earthworm populations in 6 watersheds

(Mineira, Ajuricaba, Facão Torto, Buriti, Pacurí and Toledo), in SW Paraná State, Brazil, aiming

towards a ranking of earthworm density and diversity in no-till systems. Soils in the region are

mainly Red clayey Latosols (Oxisols), derived from cretaceous era basalt flows. Earthworms were

collected in 37 farms with no-tillage aging 3 - 27 yr of age, in February 2010, using an adaptation of

the TSBF method (hand sorting of five 20×20 cm holes to 20 cm depth). In each watershed,

earthworms were also sampled in one native forest fragment, used as a control. In the 37 no-till

systems, earthworm abundance ranged from 0 - 24.2 individuals/hole (0 - 605 indiv./m2) and in the

forests, from 0.2 – 11.4 indiv./hole (5 - 285 indiv./m2). Most of earthworms encountered were exotic

species of the genus Dichogaster (D. saliens, D. gracilis, D. bolaui and D. affinis, Acanthodrilidae

family) and native Ocnerodrilidae (mainly Belladrilus sp.), all of small individual size. In a few sites,

individuals of the native Glossoscolecidae (P. corethrurus, Glossoscolex sp., Fimoscolex sp.) and

exotic Megascolecidae families (Amynthas gracilis) were also encountered, in low densities.

Urobenus brasiliensis (glossoscolecid) was found only in the forest fragments. Diversity per site

ranged from 1 to 6 sp. and the totals per watershed were: Mineira, 4 sp.; Ajuricaba, 8 sp.; Facão

Torto, 4 sp.; Buriti, 7 sp.; Pacurí, 6 sp.; and Toledo, 8 sp. In the forests, diversity was, 1, 4, 4, 2, 2,

and 6 sp. per watershed, respectively. Based on this study and results from previous studies by the

authors, a ranking of earthworm abundance and diversity in no-till systems is here proposed for the

red Latosol, warmer climate regions (Cfa, Koeppen) of Paraná State: poor = 2 indiv./hole and 1 sp.;

moderate = > 2 to 6 indiv./hole and 2 sp.; good = > 6 to 10 indiv./hole and 3 sp.; excellent = > 10

indiv./hole and > 4 sp.*Financed by Itaipu-Binacional

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Effect of afforestation and landscape position on the stable isotopic

signatures of soils, vegetation, and earthworms on the Isle of Rum,

Scotland, UK

Callaham Mac A.

1, Butt Kevin R.

2, Lowe Christopher N.

2

1USDA Forest Service, USA

2University of Central Lancashire, UK

The Isle of Rum, in the Inner Hebrides off the northwest coast of Scotland, is a 10,000 ha nature

preserve administered by the Scottish Natural Heritage (SNH). Historical land-use for subsistence

farming and sheep grazing, and more recent private management of the site as a hunting preserve for

red deer, have each contributed to the near total loss of native forest cover on the Island. However,

after the SNH assumed management of the site, one goal has been to restore forest vegetation to large

areas of the island. Afforestation efforts on Rum to date have consisted of experimental plantings of

different tree species in fenced areas at various landscape positions across the island. Previous

sampling for earthworms in these plots revealed that earthworms were found in much greater

abundances under tree cover than in open moorland. In an effort to understand how increasing cover

of forest vegetation and associated changes in soil faunal communities might be expected to

influence nitrogen dynamics, we sampled soil, vegetation and earthworms for stable isotope analysis

from beneath different tree species in several of the experimental forest stands as well as from older,

well established woodlands surrounding the village of Kinloch (the only continuously inhabited

human settlement on the island). Several patterns emerged in delta-15

N signatures of soil, vegetation,

and earthworms, with clear differences between tree species and elevation. Samples collected at

higher elevations were less enriched with 15

N than those at lower landscape positions, and soil and

vegetation collected under pines (Pinus sylvestris) was less enriched than that collected under oaks

(Quercus robur). We also observed inter-specific differences in delta-15

N signatures for earthworms

at a given site, as well as intra-specific differences in delta-15

N in earthworms collected at different

landscape positions, and in earthworms collected under different tree species. 15

N signatures were

always lowest in vegetation, and bulk soil usually 1.5-2.5‰ enriched relative to vegetation. When

Aporrectodea caliginosa were collected they were always more enriched than bulk soil, and

earthworms in the genus Lumbricus were always depleted in 15

N relative to bulk soil. Dendrobaena

spp., Dendrodrilus rubidus, and Octolasion cyaneum had variable delta-15

N tissue signatures relative

to bulk soil. These results will be interpreted in terms of earthworm feeding behavior, and nitrogen

biogeochemistry.

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Invasion of Asian Amynthas species and increase of earthworm abundance

in Mid-Atlantic deciduous forests of the USA during 1999-2010

Chang Chih-Han

1, Szlavecz Katalin

1, Pitz Scott

1, Csuzdi Csaba

2, McCormick Melissa

3,

Filley Timothy4

1Johns Hopkins University, Baltimore, USA

2Natural History Museum, Hungary

3Smithsonian Environmental Research Center, USA

4Purdue University, USA

Over 90% of the forests in the Mid-Atlantic USA are secondary forests at different successional

stages. Soil macrofauna in these forests is often dominated by cosmopolitan earthworms, while

native species are still present. In 1999 we surveyed the earthworm fauna at the Smithsonian

Environmental Research Center (SERC), Maryland. We recorded eight species in the forests,

including Allolobophora chlorotica (Savigny, 1826), Aporrectodea caliginosa (Savigny, 1826),

Aporrectodea rosea (Savigny, 1826), Eisenoides loennbergi (Michaelsen, 1894), Lumbricus friendi

Cognetti, 1904, Lumbricus rubellus Hoffmeister, 1843, Octolasion cyaneum (Savigny, 1826) and

Octolasion lacteum (Orley, 1881). Four additional species, Bimastos palustris Moore, 1895,

Dendrobaena octaedra (Savigny, 1926), Dendrodrilus rubidus rubidus (Savigny, 1926) and

Diplocardia caroliniana Eisen, 1899, were found only in non-forest habitats. Earthworm abundance

was significantly higher in young (70 yr) than in old (150 yr) forests. Species composition was also

different. In addition, smaller, uncut forest stands had no earthworms. The objective of this study was

to assess if this pattern holds ten years later. Between 2006 and 2010 we re-sampled the original plots

and added new ones. Earthworms were sampled using formalin extraction from three randomly

selected 50 cm X 50 cm quadrates at each plot. In addition to the eight species recorded in 1999 two

cosmopolitan species, Lumbricus terrestris Linnaeus, 1758 and Amynthas hilgendorfi (Michaelsen,

1892) were found in the forests. Another exotic earthworm, Amynthas hupeiensis (Michaelsen, 1895)

was newly recorded in the grassy areas. Species compositions in old forests differ from that in young

ones by the presence of the native E. loennbergi and the absence of Ap. rosea, Al. chlorotica, and O.

lacteum. Earthworm densities were significantly lower in old forests than in young ones in 2006, but

subsequent sampling revealed an increase in overall abundance. The difference between young and

old forests is diminishing. The uncut sites are still earthworm-free. Densities were lowest in the

summer with the lowest ratio of mature (clitellate) to total individuals and the possible absence of Ap.

caliginosa. We conclude that the earthworm fauna in the SERC forests is stable in terms of

community composition but dynamic in terms of abundance. The presence and possible

establishment of the three newly recorded species needs to be monitored.

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Integration of soil biodiversity in soil quality monitoring: earthworm

results of RMQS BioDiv program

Cluzeau Daniel

1, Pérès Guénola

1, Guernion Muriel

1, Cannavacciuolo Mario

2, Bellido Alain

1,

Rougé Laurence1, Boulonne L

3, Villenave Cécile.

4, Piron Denis

1


2ESA, Angers, France

3INRA UMS InfoSol, France

4IRD Montpellier, France

A French program ―RMQS BioDiv‖ was developed in Brittany (Western France) in order to classify

at a regional scale soil biodiversity in relation to land use and pedoclimatic parameters. The sampling

area covered 27 000 km² and 109 sites were studied, in the Spring of 2006 and 2007, using a

systematic sampling (regular grid 16 km x 16 km). The strength of this program was to propose a

large panel of geological and pedological contexts combined to land uses (pastures, cultures,

rotations). A large national research network (12 research teams) supported this program that

undertook the study of several biological parameters such as macrofauna (earthworms, total macro-

invertebrates), mesofauna (acarina and collembola), microfauna (nematodes) and other

microorganisms (microbial biomass, community structure and functional genes). This program was

connected to another soil monitoring network ―RMQS‖ which covered the whole French territory

and monitored physicochemical soil parameters and land-use management. The link between these

two programs, working on the same sites, permitted the study of biological parameters in the light of

soil and agricultural characteristics, and thus the definition of biological descriptors of soil quality. In

this work we will especially focus on earthworm results of the RMQS BioDiv program. First, we will

present abundance (global and by ecological categories), biomass, richness and diversity index

results. These parameters were placed into a large dataset and have the advantage of geographical

illustration at regional scale. The species assemblages will be also described thanks to the 23

earthworm species sampled in the area. Then, the relationship between earthworm parameters and

explanatory variables such as land use and agricultural practices will be put in evidence. All

combinations will be summarized in a table, in which we can distinguish, for example, that tillage

practices greatly influence anecic abundance regardless of the pedological context. Moreover, spatial

analyses conducted on earthworm parameters and species distribution will be shown. Actually, few

species (e.g. Lumbricus terrestris, Aporrectodea nocturna) displayed spatial patterns at a regional

scale. Finally, links existing between earthworm and other biological groups sampled in the RMQS

BioDiv program will be shown, especially the significant co-structure that exists between nematodes

and earthworm communities.

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The need for meta-databases to store and manage soil biological data

Cluzeau Daniel

1, Rougé Laurence

1, Cannavacciuolo Mario

2, Bellido Alain

1, Jolivet C.

3,

Guernion Muriel1, Briard Charlène

1, Piron Denis

1, Pérès Guénola

1


2ESA, Angers, France

3INRA UMS InfoSol, France

One of the eight major threats on soil identified in the EU‘s Thematic Strategy for Soil Protection

(COM 2002, 2006) is the decline in soil biodiversity. Moreover, the pertinence of some biological

groups as indicators is now largely admitted, and the ―top 3 indicators‖ have been defined during the

ENVASSO Program as the minimum set of indicators for assessment of biodiversity decline: 1)

Earthworm diversity and biomass; 2) Collembola diversity; and 3) Microbial respiration. In order to

study soil biodiversity (species and function), numerous long term monitoring programs are being

carried out at different scales (local, regional, national, European and International scale) and on

various agricultural systems by the EcoBio team of the University of Rennes (France). Research

mainly addresses earthworm communities in regard to soil parameters and agricultural practices, and

their relationship to the other biological groups, from microbes to macrofauna. Since vast data sets

are generated by these different programs, and in order to facilitate data analysis, it became evident

that an efficient tool of storage and management was required. Thus, since 2007, a data base model

has been developed. Originally dedicated to a regional program ―RMQS BioDiv‖ which produced

more than 80 000 data points, it has been progressively adapted to be compatible to store data from

all soil biodiversity monitoring studies and resulted in the creation of a relational meta-database. It

consists of standardized modules, each module storing data from a particular program. Moreover, the

meta-database was designed to be interoperable with other databases for instance with the national

DONESOL database (Infosol INRA, Orléans), thus permiting analysis of earthworm data in relation

to soil physicochemical data. Furthermore, it is interoperable with the Lombricien2000 database of

the CNUSC (INRA, Montpellier) which contains all the data generated by the studies of Marcel

Bouché since the 1960s, including anatomo-morphologic descriptions of earthworm species as well

as data from pan-European inventories. A user-friendly interface has been designed, and will be soon

accessible over the web, stimulating information sharing and the building of a real biodiversity

observation system. The aim of such a poster is to present the structure of this meta-database, its

components and functions, and examples of possible applications.

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Use of earthworm traits to understand links between species assemblages

and their environment

Cluzeau Daniel

1, Guernion Muriel

1, Briard Charlène

1, Rougé Laurence

1, Qiu Jiang-Ping

2,

Pérès Guénola1


2Shanghai Jiao Tong University, China

Species‘ functional characteristics strongly influence ecosystem properties. Functional characteristics

operate in a variety of contexts, including effects of dominant species, keystone species, ecological

engineers and interactions among species. Most studies in community ecology are based on species

diversity and relative abundance but these parameters alone are not always a good predictor of

species importance at the ecosystem-level. Recently, numerous studies are attempting to use

functional traits to link global change drivers, functional diversity and ecosystem processes, in order

to develop indicators or generalize ecological patterns. An understanding of how changes in species

richness and composition, and biodiversity in general, influence ecosystem properties requires

knowledge of the functional traits of the species involved. By definition, functional traits are those

that influence ecosystem properties or species‘ responses to environmental conditions. Many studies

have addressed the link between plant species traits and their environment, but few studies have

involved soil fauna. In this work we focus on temperate earthworm species, because as soil

ecosystem engineers, they play an important role in, and interaction with their environment. In this

context, our aim was to determine which environmental variables control community assemblages

using earthworm traits. To reach this aim, we used data stored in the Lombricien2000 database.

These data include earthworm taxa and environmental variables collected in France by Marcel

Bouché (1972). Environmental variables included C, N, pH, Ca, altitude, soil texture and land use

from about 1000 sites. It included artificial surfaces, agricultural areas, forest and semi natural areas.

Species life history traits were selected among anatomical and morphological characteristics (e.g.

calciferous glands, bladder shape, typhlosole, body size, body shape, pigmentation, resistance form).

We used a three-table ordination method (RLQ analysis) to identify the relationship between

environmental variables and species traits. Results demonstrated a gradient within the environmental

data (C and N opposed to pH and Ca). The relevance of anatomical and morphological traits used to

link community structure and environmental variables will be presented and discussed.

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Earthworm community response to a disturbance gradient

Falco Liliana

1, Momo Fernando

2, Sandler Rosana

1, Coviella Carlos

1

1Universidad Nacional de Lujan, Argentina

2Universidad Nacional General Sarmiento, Argentina

The aim of this study was to determine the earthworm community response to a soil deterioration

gradient. The gradient was determined according to different management practices as follows: 1) a

system with 40 years of uninterrupted agriculture, under no tillage practice during the last 16 years,

2) a 16 year old grazing field under feedlot management since two years ago, and 3) an abandoned

pasture that has not been disturbed during the last 50 years. The samples were taken every three

months between August 2008 and August 2009. For each system, five samples 25 x 25 x 25 cm were

taken. Numbers of individuals and species composition were measured. Physical, chemical, and

microbiological variables were measured at the same sampling points. The relation between these

variables and earthworm presence was analyzed. Usage history showed differences between

variables, through discriminant analysis. These differences were mirrored by earthworm species

composition and abundance. A Mann-Whitney analysis (p< 0.05) showed that earthworms preferred

environments with P levels higher than 20 ppm, Ca levels higher than 11 meq/100g, K below 1.4

meq/100g, total N content above 0.25%, soil humidity above 15%, low compaction values (<1,2

g/cm3) and low mechanical resistance. Species distribution was, in turn, associated to some of the

variables mentioned. In this way, the abandoned pasture had soils with neutral pH, the lowest levels

of K, was slightly compacted and had high activity of N2-fixing bacteria. In these soils the native

Microscolex dubius was dominant, with the presence of Aporrectodea and Octolasion sp. The long-

term agricultural system had slightly acidic soils, high Ca and Mg, intermediate K level, low total N

and low activity of N2-fixing bacteria. In these soils, the exotic Aporrectodea was the dominant

genus, followed by Octolasion and Micrsocolex. The old grazing field was different from the

agricultural system by having lower P, Ca, and Mg, and high levels of K and total N. However,

earthworm presence in this environment was associated to high soil humidity, with the native

Eukerria stagnalis present, indicating flooding problems not detected by the chemical and physical

analyses. Even though the analyzed variables showed statistically significant differences, from an

agricultural point of view (INTA agronomic indicators) the systems did not differ from each other

nor showed signs of deterioration. But earthworm species composition and abundance indicated

present environmental conditions and the risk of resource deterioration.

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Diversity and abundance of earthworms in land use systems in Central-

Western Colombia

Feijoo Alexander

1, Carvajal Andrés F.

1, Zúñiga Maria C.

1, Quintero Heimar

2

1Universidad Tecnológica de Pereira, Colombia

2Universidad Nacional de Colombia, Colombia

The Neotropical landscape of the Colombian Andes is characterized by a mosaic of agroecosystems

with diverse land cover in which the adaptation ability of soil fauna is still unknown. To analyze the

diversity and abundance of earthworm species in relation to changes in selected physical and

chemical parameters (bulk density, C & N stocks, %C, %N, 13

C, 15

N, C:N ratio) the systems were

classified in five categories according to land-use intensity: non-intensive (NI), low intensity (LI),

medium intensity (MI), high intensity (HI) and highest intensity (IN). There were significant

differences (p<0,05) in C and N contents of IN systems (pastures), compared to the other categories.

In total, 26 earthworm species were recorded of which 16 showed a high adaptation capacity to

natural (NI) and to low and medium disturbance (LI, MI) systems. Depending on the use intensity,

there were also significant differences (p<0.05) in the origin (native vs. exotic) and ecological

category of earthworms, mainly in the variables native species diversity, endogeic species diversity,

native species abundance, native species biomass, and exotic species abundance. The results allow

deduction of which mosaic systems are more favourable for agrobiodiversity conservation and for

which subsystems special management is required to resolve problems of habitat degradation.

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Impact of floodplain restoration on earthworm communities

Fournier Bertrand, Samaritani Emanuela, Le Bayon Claire, Mitchell Edward A.D.

University of Neuchâtel, Switzerland

Floodplains have been intensively embanked, dammed or otherwise engineered in industrialized

countries, but are now increasingly being restored, often at high cost. Increasing biodiversity is a

major goal of these projects. There is however much confusion over the impact of such

transformation on below-ground communities. Even a functionally essential group such as

earthworms remains understudied in this context. Using a functional process zones (FPZ) approach,

we aimed at assessing the differences along a perturbation gradient (fluvial dynamic) of a recently

restored floodplain in terms of earthworm diversity and community structure, defining the relations

among earthworms and ecosystem functions and among earthworms and other soil fauna

communities. The study took place along the Thur River in western Switzerland. The site was

divided into six FPZ showing different fluvial dynamics. Six replicates were used in the most

dynamic ones (from gravels to willow bushes) and four in the most stable ones (forest). A non

restored section of the river (pasture) was sampled as a pre-restoration control. Site (coordinates,

elevation, vegetation structure and soil morphology) and functional (litter input and decomposition

rate, C and N cycling, enzymatic activity, microbial biomass and soil respiration) variables were

measured. Earthworms were sampled in early October 2008 by mustard powder extraction. Their

functional role was assessed by calculating the fraction of variance explained by each explanatory

variable. The relations with other communities (vegetation, spiders, testate amoebae and bacteria)

were investigated using multiple factor analysis. Earthworm diversity and species richness increased

along the gradient. The highest values were obtained far from the river in the most stable FPZ. The

composition of earthworm communities differed between the dynamic and stable FPZ and between

the restored and control areas. This pattern was best correlated to litter input, lateral distance to the

river and plant community composition. Restoration changed earthworm communities that are now

structured into FPZ. Litter input is of primary importance for this pattern. As a result, changing plant

communities may change the quality and quantity of litter inputs as well as the related earthworm

communities which may in turn influence organic matter cycling. This highlights the importance of

below-ground communities for the success of restoration projects.

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Passive invasion of earthworms in rehabilitated open mined soils: the

effect of time, topography and vegetation types

Fragoso Carlos, López Valeria, Angeles Antonio

Inecol, A.C. Mexico

The extraction of primary materials in open cement mines eliminates the entire soil biotic

community. To recover ecosystem function and biodiversity, rehabilitation and restoration practices

include soil inoculation from neighbouring or foreign sites, the introduction of different plant types

and other ecological strategies. In this study we evaluated the passive invasion (no inoculation

implemented) of earthworms in open cement mined soils in central Mexico, rehabilitated 17 years

ago with four main strategies/treatments: slope with trees (ST), slope with grasses (SG), flat clay

banks with trees (FT) and flat clay banks with grasses (FG). Earthworms were sampled in 1995 (2

years after rehabilitation), 1998 (5 years after) and in 2009 (16 years after). Control sampling of

earthworms was made in a nearby tropical undisturbed forest. Our results indicate that species

richness, abundance and vertical distribution increased with time, and that some natives were capable

of re-colonizing rehabilitated soils. In some treatments, species richness and abundance were even

higher than in tropical forests; vertical distribution in all treatments, however, was always more

superficial than in forests.

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Earthworm abundance and biomass in native Brazilian Atlantic forest and

Araucaria angustifolia and Pinus elliotti plantations collected using two

sampling methods

Godoi de Lima Odair

1, Da Silva Elodie

2, Piacentini de Andrade Daniel

3, Brown George

4

1Universidade Federal do Paraná (UFPR), Brazil

2Université Paris-Est Créteil, France

3Faculdades Integradas Espírita (Unibem), Brazil


Earthworms are important indicators of environmental quality and management, such as the

conversion impacts of native to planted forests. Nevertheless, little research has been conducted in

this field in Latin America. Therefore, the present work was undertaken to determine the effect of

native and exotic tree plantations on earthworm populations in the Atlantic Forest region of Southern

Brazil. Earthworms were sampled in sixteen areas at the Embrapa Forestry Research Station in

Colombo, Paraná State, Brazil, including five areas with Ombrophyllous mixed forests (Atlantic

forest with native Araucaria angustifolia trees), five A. angustifolia plantations and six Pinus elliotti

plantations (PP). All the Araucaria and Pine plantations were established between 1979-1987. Two

sampling methods were used: a) excavation of 40 × 40 cm monoliths and manual sorting of the soil at

the depths of 0-10 and 10-20 cm, and b) application of 20-L of diluted formaldehyde (0,5%) on an

area of 1m2. Earthworm species density and biomass obtained with each sampling method were

assessed and compared across the areas. The most abundant species were Pontoscolex corethrurus, a

peregrine species and Amynthas gracilis, an exotic. A few individuals of Amynthas corticis and

Metaphire schmardae (exotics) and of the native species Urobenus brasiliensis and Andiorrhinus sp.,

were also found. The highest abundance of earthworms was encountered using hand-sorting in Pine

plantations (116 ind/m2), followed by the Araucaria plantations (64 ind/m2) and the lowest

abundance was found in the native forests (38 ind/m2). On the other hand, biomass was similar in the

Araucaria and Pine plantations (around 25-26 g/m2), and the lowest biomass was found in the native

forests (12 g/m2). With the formaldehyde sampling, earthworm abundance was much lower, ranging

from 11 (Araucaria plantations) to 20 ind/m2 (native forests). Biomass ranged from 6.6 (Araucaria

plantations) to around 9 g/m2 (native forests and Pine plantations). Therefore, although all species

were collected using the formaldehyde method, hand-sorting for earthworms was much more

efficient for extracting earthworms from the studied forest ecosystems.

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Diversity of earthworm communities as indicators of flood regime on

coastline

Ivask Mari, Kuu Annely

Tallinn University of Technology, Estonia

The salinity of sea water, periodical flooding and dry-off of habitat as well as unfavourable soil

texture has strong negative impacts on earthworm communities. In spite of this, most Estonian

earthworm species are present on the coast of the Baltic Sea and the community composition can be

used as indicator of flood regime. The grasslands of the Western Estonian coast are on saline littoral

soils. Distribution and abundance of earthworms are influenced by flooding duration and character.

The salinity in the Baltic Sea water is low (6-8‰), the salinity of Matsalu and Liivi Bays being lower

(5-6‰) or nearly fresh at the river mouths. The brackish conditions of the Baltic Sea create special

habitats for earthworms. We studied earthworm communities on the coast of Matsalu Bay and Liivi

Bay, in different zones divided on the basis of degree of influence of the sea and characterized by

plant species that tolerate various levels of salinity and humidity. In Estonia 13 species of

earthworms have been founded so far, all peregrine cosmopolitans. Earthworm abundance on the

coastal areas ranged from 0 to 84 individuals m-2. Epigeic species are more tolerant to limiting

factors and they recolonize coastal areas very quickly after unsuitable conditions. Endogeic species

are more sensitive to submersion and unfavourable soil texture. Only the semiaquatic earthworm

Octolasion lacteum was able to inhabit coastline areas mostly flooded by seawater. Coastal areas are

impossible habitats for anecic species because of high groundwater table. The most tolerant species

to extreme conditions was Lumbricus rubellus being dominant in the subsaline zone of the coast.

This is generally an area underwater that dries up when water level decreases. Here O. lacteum was

also abundant. Epigeic earthworms, Dendrobaena octaedra, Eiseniella tetraedra, Dendrodrilus

rubidus and Lumbricus castaneus were present in both subsaline and saline zones of the coast. In the

suprasaline zone a small number of Aporrectodea caliginosa and A. rosea were present on areas

where the soil layer was developed. Five peregrine species (endogeic and anecic) were not present in

the zones under influence of sea water. In conclusion, we found five epigeic and three endogeic

species living in habitats with periodic flooding by brackish sea water. These peregrine species were

able to recolonize the habitats on the coast after flooding and can be used as indicators of changes in

salinity and flood character of coastal areas.

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Impact of flood deposits on earthworm communities, humus forms and soil

parameters in alder forests from a subalpine floodplain (Kandersteg,

Switzerland)

Le Bayon Claire

1, Bullinger-Weber G.

2, Guenat C.

3, Salomé Clémence

1, Gobat J.M.

1

1University of Neuchâtel, Switzerland

2Univeristy of Lausanne, Switzerland

3Ecole Polytechnique Fédérale de Lausanne, Switzerland

Floodplains are areas of land adjacent to rivers and streams that are subject to recurring inundations.

As a result of this dynamic, vegetation changes along a gradient of flooding frequency and duration,

and usually consists of many small patches of vegetation with different species composition and

succession of stages often related to fluvial landforms. Regarding soils, they are highly variable and

also strongly related to fluvial dynamics; the coarsest sediments being deposited on the natural levee,

immediately adjacent to the stream channel, while finer soil particles are progressively deposited

with increasing distance from the stream. In this context, we studied a floodplain at the subalpine

level (1200 m) that presents visually a flow-rate gradient over approximately 3 km. We hypothesized

that biological and physicochemical soil parameters varied along this river gradient. In order to

eliminate the variation of vegetation, and because forests of alder shrubs and trees were recovered

from the highest flow regime to stagnant waters, we described humus forms and soil types in seven

alder phytocenoses. Soil samples were removed at two depths when possible (0-10 and >10 cm) and

extraction of earthworms was performed at each of the seven stations. Soil analyses included pH

(water and KCl), cation exchange capacity (CEC), amount of organic matter (loss on ignition, carbon

and nitrogen), amount of CaCO3 (total and active) and soil texture. Fungal mycelia was also

qualitatively observed. Earthworms were weighed and identified to species level. First results

demonstrated a high variation of soil organic matter and active CaCO3 in the first 10 cm, as well as a

great difference in soil texture. Thus, at the soil surface, coarse sands were positively correlated with

high flow regimes, while the inverse tendency was observed for fine loam and clays. No strong

variation was observed for pH, CEC and total CaCO3. Studies on the earthworm communities are

still in progress, as well as humus forms and total C and N.

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Invasion of Pontoscolex corethrurus (Glossoscolecidae, Oligochaeta) in

Amazonian landscapes

Marichal Raphael

1, Feijoo Alexander

2, Praxedes Catarina

3, Ruiz Dario

2, Carvajal Andrés F.

2,

Oswald Johan4, Hurtado Ma. Del Pilar

5, Brown George

6, Grimaldi Michel

7, Desjardins

Thierry7, Sarrazin Max

7, Decaëns Thibaud

8, Lavelle Patrick

7

1Université Pierre et Marie Curie and IRD, France

2Universidad Tecnológica de Pereira, Colômbia

3Museu Paraense Emilio Goeldi, Brasil

4Université de Rennes, France

5CIAT, Colombia

6Embrapa Florestas, Brasil

7Centre IRD Ile, France

8Université de Rouen, France

Pontoscolex corethrurus (Glossoscolecidae, Oligochaeta) is an invasive endogeic earthworm that has

colonized most land transformed by human activities in the humid tropics. When installed,

populations can change soil physical properties, biogeochemical processes and microbial

communities. The aim of this study was to determine whether P. corethrurus establishment is a result

of: 1) a competitive exclusion of native species, or 2) the exploitation of a new niche created by

disturbance that native species cannot use. We tested these hypotheses by performing a survey of

earthworm communities in 270 sample points that covered the diversity of land use systems

encountered in two contrasted regions of the Amazonian arc of deforestation, in Brazil and

Colombia, respectively. Independence between the processes that determine each group density,

indicating an absence of competitive exclusion, was verified by their respective locations on

orthogonal (Colombian points) or quasi-orthogonal (Brazilian points) axes of a Principal Component

Analysis of their abundance across sampling points and land uses. Pontoscolex corethrurus densities

co-varied with soil N content and pH (all points). Our results thus suggest the latter hypothesis, i.e.,

that P. corethrurus, unlike native species, is able to feed and develop in environments where litter

resources are severely decreased and soils have been enriched in C and nutrients by deforestation and

burning.

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Dew worms in the white nights

Nuutinen Visa

1, Butt Kevin R.

2, Shipitalo Martin J.

3, Sirén Taisto

1

1MTT, Finland

2UCLan, UK

3USDA-ARS, USA

Lumbricus terrestris L. (the dew worm) forages, mates and migrates on the soil surface during the

night. Its distribution covers a broad latitudinal gradient and variation in day length conditions. Since

soil-surface activity is crucial for the survival and reproduction of dew worms, it is conceivable that

northern populations that experience light nights in the summer have adapted to remain surface active

in twilight and show less sensitivity to light compared with more southerly populations. We

evaluated this hypothesis experimentally. Adult dew worms were collected from a wide latitudinal

gradient in May 2009, from field sites in Jokioinen (Finland; 60º48‘N, 23º28‘E), Preston (UK,

53º46‘N, 2º42‘W) and Coshocton (USA, 40º16‘N, 81º50‘E). In Jokioinen there is a 3-week period

without a dark-night interval (twilight prevails throughout the night) in the middle of summer, while

in Preston even the shortest midsummer night has 5 hours of darkness and, respectively, 8 hours in

Coshocton. Using field-collected animals, an experiment was conducted in Jokioinen from June 6 to

June 21 - midsummer. Groups of three individuals from a particular geographic origin were housed

in soil-filled PVC-tubes. Before the experiment they were kept in darkness and then under an

artificial light-cycle to equally distort any inherent daily activity pattern. Three replicate tubes were

exposed to either ambient natural light or artificially induced darkness during the night. Earthworm

surface activities were recorded from above using low-light video cameras from 22:30 to 04:30 hrs,

which covered the period between sunset and sunrise. The main response variable was surface

activity (activity vs. no activity in 0.5 hr increments). Preliminary analysis of the data has shown that

under artificial darkness, surface activity was high throughout the night for all three dew worm

origins. Under ambient light, surface activity increased gradually after sunset, peaked after midnight

and then declined towards sunrise. Giving tentative support for the initial hypothesis, under natural

light Finnish earthworms began their surface activity earlier than the others and remained active for

the longest period. In the statistical analyses currently underway, we are using air temperature and

moisture together with an index of cloud cover as covariates. Our presentation will report the full

results and discuss their importance and implications.

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Tillage practices and organic fertilization influence endogeic earthworms

Piron Denis, Pérès Guénola, Moussay Mickaël, Guernion Muriel, Bellido Alain,

Cluzeau Daniel

UMR EcoBio CNRS_Univ-Rennes1, France

In agricultural soil, it is widely recognized that reducing soil tillage and organic fertilization can,

independently, improve earthworm communities. On one hand, conservation tillage management

promotes anecic species thus increasing total earthworm biomass. On the other, organic fertilization

improves the whole earthworm community. However, the response of earthworm communities to a

combination of reduced tillage and organic fertilization is less well documented, despite the

potentially favourable aspects of this agricultural management system. Our study was based on an

experimental site in Britain (France) that combined three tillage managements: conventional tillage

with plough (CT); superficial tillage without plough (ST) and No tillage (NT), with mineral

fertilization (M), poultry manure (PM) and Pig slurry (PS). After eight years, earthworm

communities were sampled by formaldehyde extraction. Complementarily, earthworms were

sampled at different depths (0-5 cm; 5-10 cm and 10-20 cm). In mineral fertilization, ST (102 ind.m-

2) and NT treatments (196 ind.m-2) were more favourable to earthworm abundance (and biomass)

compared to CT treatment (62 ind.m-2). With organic fertilization, this trend was also verified in

term of biomass, but not in term of abundance. Indeed, anecic earthworms were supported by the

reduction of tillage (increasing total earthworm biomass) whereas endogeic earthworms were

supported by organic fertilization (increasing total abundance). Therefore, endogeic abundance reacts

differently to soil tillage reduction according to organic fertilization. Although no significant

difference was observed, superficial tillage (ST) affected more endogeic species (91 ind.m-2)

compared to CT (121 ind.m-2) with pig slurry; abundance being equivalent in CT (165 ind.m-2) and

ST (152 ind.m-2) with poultry manure. In conventional tillage (CT), endogeics were regularly

distributed on the whole ploughed layer (0-20 cm) whereas they were concentrated in the first 5

centimetres in ST and NT. This study indicates that the superficial tillage can be as harmful as

traditional ploughing because endogeic species live near the soil surface and are more susceptible to

superficial soil disturbance. Just like reduced soil tillage, organic fertilization appears to be a good

practice for soil restoration, but acts differentially on the functional component of the earthworm

community.

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The effects of production systems on earthworm assemblages in vineyards

and apple orchards

Pizl Vaclav

Biology Centre ASCR, Czech Republic

Three different production systems in vineyards and apple orchards, i.e. biological (BP), integrated

(IP) and conventional (CP), were compared for their effects on earthworm communities. Commercial

vineyards and apple orchards were studied in three wine and three fruit production areas in the Czech

Republic. In each area, plots under BP, IP and CP, and a nearby natural site (control), were chosen

for the study. In spring and autumn 2009, soil samples were taken in two vine rows or tree lines and

adjacent alleys of each plot and in transects across natural sites. Earthworms were extracted by heat

using a modified Kempson apparatus. In total, 10 earthworm species were found, 6 in vineyards and

7 in orchards. In nearby natural sites 8 species were recorded and 3 of them were exclusives. The

average species number per-site was higher in BP and IP than CP vineyards and in BP than in IP and

CP orchards. The community structure in IP vineyards and in IP and CP orchards was similar in all

production areas, while those in BP vineyards and orchards differed strongly between production

areas. Earthworm density and biomass were higher in IP than in CP vineyards, whereas both

parameters in BP did not differ significantly from those in IP and CP vineyards. In apple orchards,

the earthworm density was higher under CP and IP than under BP. However, there was no difference

in earthworm biomass among differently managed orchards. In vineyards, earthworm density and

biomass were significantly lower in the vine rows than between them. In contrast, both parameters

were higher in the tree lines than in the alleys of orchards. The results are discussed with reference to

management practices (soil tillage, mulching and pesticide applications) used in plots under different

production systems.

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Edaphobase – an online soil organism data warehouse for taxonomy,

literature and ecological data management: The example of earthworms

Roembke Joerg

1, Jaensch Stephan

1, Scheffczyk Adam

1, Volkmer Louise

1, Russell David

2

1ECT Oekotoxikologie GmbH, Floersheim, Germany

2Senckenberg Museum of Natural History, Görlitz, Germany

Edaphobase is a database project aiming to bring together information concerning taxonomy,

literature references and ecological requirements of important soil taxa in a combined database sys-

tem. At present, Collembola, Enchytraeidae, Gamasina, Lumbricidae, Myriapoda, Nematoda and

Oribatida will be included. Data are to be extracted from literature, collection data and unpublished

material from research institutions with a focus on Germany and its neighbouring countries. Linking

such data on a wide range of soil-organism groups from several trophic levels with climatic, habitat

and geographical data will allow detailed subsequent analyses on geographical distribution as well as

ecological requirements of single species such as, e.g., preferences for temperature, pH, moisture or nitrogen content or effects of anthropogenic perturbation. In addition, prognostic tools

will be provided to discern changes within soil biocenotic assemblages as a reaction to, e.g.,

modifications in land-use techniques or effects of climatic changes. The final data set will be

available for public access via a web-based application, including investigations tools for

construction of fine-scaled distribution maps, analysis results regarding parameter preferences or

estimations of threats to soil-organism distribution. First experiences with earthworms are

presented and discussed.

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Ecological stoichiometry in earthworms (Oligochaeta, Lumbricidae)

belonging to different ecological groups

Rozen Anna, Sobczyk Lukasz, Weiner January

Jagiellonian University, Poland

The growing interest in applying stoichiometry approaches to community ecology has resulted in

numerous studies concerning aquatic systems, while terrestrial (soil) biota are still poorly known in

this respect. Usually, species closely related taxonomically and belonging to the same functional

group may be expected to have similar stoichiometric body composition. On the other hand, from

ecological niche theory, we can expect that closely related species of soil fauna coexisting in

relatively homogenous environments should realize different niches that may be reflected in different

stoichiometry. Conversely, species characterized by similar stoichiometry should be competitively

excluded. The aims of our study were to check: (i) if earthworms belonging to various ecological

groups differ in body composition and stoichiometry, (ii) if earthworms belonging to the same

ecological groups are homogenous with respect to body composition and stoichiometry. The study

was conducted on earthworms belonging to three different ecological groups: anecic (Lumbricus

terrestris), endogeic (Aporrectodea rosea) and epigeic (Dendrobaena octaedra, Dendrodrilus

rubidus). Contents of macroelements (C, N, S, P) and microelements (Mg, K, Ca, Fe, Cu, Mn, Zn)

were analyzed in earthworms bodies, using the following methods: C, N and S with CHNS analyzer,

P colorimetrically using molibdenian blue method (FIA), and Mg, K, Ca, Fe, Cu, Mn, Zn by AAS

using standard procedures. We found that in earthworms the mean concentration of macroelements

was: C 45.9%, N 10.2% and P 1.1%. The ratio C/N was 4.5, C/P 40.3 and N/P 9.0. The average

contents of microelements in earthworms were: Ca 9664.4 mg/kg, Zn 284.7 mg/kg, Mn 107.8 mg/kg,

Mg 695.4 mg/kg, Fe 564.9 mg/kg, Cu 22.9 mg/kg, and K 9111.1 mg/kg. We found significant

interspecies differences in body composition. D. rubidus differed from D. octaedra in the N content,

and D. rubidus differed from D. octaedra and L. terrestris in C content. Interspecies differences were

also been found in the content of Ca, Zn and Mn, while the concentration of P was similar in all

species. The obtained results are discussed.

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Soil-dwelling annelids during meadow restoration on arable soil using

different restoration schemes

Schlaghamersky Jiri

1, Pizl Vaclav

2

1Masaryk University, Czech Republic

2Biology Centre of the Academy of Sciences, Czech Republic

Earthworms, enchytraeids and other annelid taxa present (Rhyacodrilinae; Aeolosomatidae) were

studied during a field experiment comparing several approaches to meadow restoration on arable soil.

The study site was situated in the White Carpathians (Czech Republic), where meadows, rich in herb

species and of high conservation value, have been one of the traditional land-uses, providing

protection to the local erosion-prone soils on flysch. The experiment was conducted on a former

meadow that had been used as an arable field from 1970 to 1998. The soil was luvisol (clay-loam; pH

6.0-7.0; highly variable soil moisture). In spring 1999 the site was subjected to different treatments in

a randomized block design. Our study focused on three treatments: (1) sowing with regional seed

mixture, (2) sowing with commercial grass seed mixture, and (3) natural regeneration (old field

succession directed by annual mowing). All experimental plots were mown each year in summer.

Samples were also taken in an adjacent arable field, representing the pre-restoration situation, and in

a well-developed herb-rich meadow, representing the approximate restoration target. Soil-dwelling

annelids were studied from 2000, a full sampling programme with three samplings per year covering

all replicates of the above treatments was conducted in 2002-2004, another sampling followed in

Nov. 2009. In total, 4 earthworm species were founded in the field, 7 in the restoration plots, and 8 in

the well-established meadow. Octolasion lacteum, Aporrectodea trapezoides and A. caliginosa

dominated in the initial phase of meadow restoration. A. trapezoides had almost disappeared by 2004,

A. rosea, one of two dominant species in the meadow, became dominant in all restoration plots.

Annual densities were below 7 individuals/m2 in the field, about 15 ind./m2 in the sown plots, over

40 ind./m2 in the natural regeneration plots, and 50-60 ind./m2 in the meadow. In total, 30

enchytraeid species and 3 other small annelids were identified. Their assemblage in the arable field

was impoverished, that of the meadow the richest. All assemblages were dominated by Fridericia

spp. and Enchytraeus spp. Annual densities ranged from 700 to almost 19 000 ind./m2 in the

restoration plots. Neither earthworm nor enchytraeid densities showed a trend in the course of

succession, possibly because of a drought in 2003. Enchytraeids showed a shift in vertical

distribution towards the upper 3 cm of soil during the experiment.

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Earthworm population dynamics under the impact of agricultural

practices and meteorological factors in the most common soil types in

Latvia

Ventins Janis

Faculty of Geography and Earth Sciences, University of Latvia

The data of the State Agricultural Land Monitoring program were used in the present analysis. These

data included long term observations of agricultural soil properties and analysis of earthworm

population dynamics under the impact of intensive agricultural practices and fluctuations in

meteorological factors at four monitoring sites, representing the most common soil types in Latvia:

sod podzolic sand and loamy sand soils, sod podzolic gleyic loam soils, sod calcareous loam soils

and fen peat soils. Sample plots included arable lands used for growing different crops over long time

periods. Earthworms were sampled from 1992 to 1998 every year in autumn. In total six earthworm

species were found in the study sites: Aporrectodea caliginosa, A .rosea, Allolobophora chlorotica,

Lumbricus terrestris, L. rubellus and Dendrobaena octaedra. Only two species were present in all

sampling plots: A. caliginosa and L. rubellus. In permanent pastures there was a tendency for 2-3

earthworm species associations. In fields with intensive use there was a distinct dominance of A.

caliginosa; other species were found in low numbers or not at all. The most favourable soils for

earthworms were the sod podzolic loam and gleyic loam. In general, the greater abundance of

earthworms corresponded to the more beneficial weather conditions characterized by warm and

moderate wet season. After dry and hot periods in summer, a substantial reduction in number of

worms occurred. In pastures with low intensity of soil disturbance, the impact of meteorological

factors had less effect on the year-to-year variability of the earthworm communities. Perennial

vegetation reduced the influence of unfavourable climatic factors and enabled a more even

distribution of worms in the soil. The density and composition of earthworm communities were good

bio-indicators for assessment of the agricultural soil quality. Mechanical soil disturbance like

intensive tillage significantly affected abundance and diversity of earthworms and resulted in

reduction in earthworm numbers and species diversity. In general earthworm communities including

less than 3-5 earthworm species are indicators of possible damage of agricultural soils. A

considerable factor determining earthworm populations was also relief. On the top of the hills

earthworms were usually less abundant than at the base because of more intense soil erosion. In the

depressions the ground-water level often was closer to the soil surface. During dry summer months,

this last factor is of particular importance for earthworms.

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Earthworms in agroforestry systems and regenerating Atlantic forest in

Barra do Turvo - SP and Adrianópolis – PR, Brazil

Wagner Maschio, Brown George

Embrapa - Florestas / Universidade Federal do Paraná, Brasil

Agroforestry systems (AFs) provide greater vegetation cover and soil protection, adding

larger amounts of residues and organic matter that serve as food for soil fauna. Therefore, in

this management system, populations and diversity of soil fauna tend to be higher than in

more intensive land use systems. Nevertheless, there are few studies about earthworm

communities in Brazilian AFs, and that compare populations in AFs with less intensive

systems such as regenerating Atlantic Forest. Therefore, the present study was undertaken to

evaluate earthworm populations in three AFs of 4, 8 and 16 yr of age, and in three secondary

forests of 5, 20 and >30 yr of age, following the TSBF (Tropical Soil Biology and Fertility)

program‘s standard sampling protocol. Two samples were collected in each of the three plots

established in each land use system, totaling six samples per site. Earthworms were

identified, counted, weighed and the results analysed using ANOVA‘s and principal

components to evaluate relationships with site characteristics. Only four earthworm species

were found, all exotic or peregrine: Pontoscolex corethrurus (Müller, 1857), Amynthas

gracilis (Kinberg, 1867) and two unidentified species of theDichogaster genus. The former

species dominated the earthworm communities, representing over 90% of the total density

and biomass of the study sites. A positive relationship between abundance and AF system

age was found, especially for the more abundant species (P. corethrurus). However, in the

secondary forests, the inverse relationship was found, with lower earthworm abundance with

increase in age of regeneration. Highest abundance was found in the 16 yr old AF (459

individuals m-2

) and in the 5 yr secondary forest (733 individuals m-2

); both systems had a

very high biomass of aprox. 100 g m-2

. Earthworm abundance was positively associated with

litter biomass and negatively related to soil clay and C content, bulk density, pH and base

saturation.

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Relationship between Oligochaeta and plant communities: a case study

from North-central Uruguay

Zerbino Maria Stella

Instituto Nacional de Investigación Agropecuaria, Uruguay

Community composition of terrestrial Oligochaeta depends on climatic, edaphic, topographic and

vegetations conditions. The aim of this study was to describe the relationship between plant

communities of the Basaltic region of Uruguay and earthworm communities. In the spring 2007,

plant communities present in the study area were surveyed: Meso-xerophytic grasslands (PMX, 11

sites); Lithophytic steppes (EL, 3 sites) and Meso-hydrophytic grasslands (PMH, 8 sites). We used

the standard Tropical Soil Biology and Fertility (TSBF) method that consists of hand-sorting

earthworms from soil monoliths 25 x 25 cm and 30 cm depth. In each site eight samples were taken

every 5 meters on two parallel transects of four points separated by 20 m. Oligochaeta from

Ocnerodrilidae, Acanthodrilidae and Glossoscolecidae families were collected. PMH had the highest

density of individuals (181 ind/m2) and Ocnerodrilidae was the dominant family (94%). PMX had a

density of 20 ind/m2 with Ocnerodrilidae and Glossocolecidae as the most abundant families. These

represented 49 and 42% of the total individuals, respectively. EL had a density of 50 ind/m2 with

Ocnerodrilidae (83%) being the most abundant family. In this plant community, Acanthodrilidae

(15%) was the second most important family. Results suggest a relationship between the earthworm

communities and vegetation, allowing the determination of biological assemblages.

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Session 4

Earthworms and Ecosystem Services

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Earthworm activities, soil functioning and ecosystem services

Decaëns Thibaud

Laboratoire ECODIV, EA 1293, FED SCALE, Bâtiment IRESE A, UFR Sciences et Techniques, Université de

Rouen, 76821 Mont Saint Aignan Cedex, France.

Earthworms have been described as ecosystem engineers that act as powerful regulators of soil

functioning at different scales of space and time. At a local scale, they produce large amounts of

organo-mineral structures (burrows, chambers and casts) that have specific impacts on soil physical,

chemical and biological processes. At higher scales, their aggregated effects may significantly impact

ecosystem functioning and the providing of important environmental services for human societies.

Here I present a state of the art review of what is currently known about the impacts of these

organisms on soils and how these impacts may affect services delivering by terrestrial ecosystems.

The approach considers several observation levels, allowing the scaling of processes from the species

functional domain to their impacts at the ecosystem scale.

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Oral – Session 4

Anecic earthworms in New Zealand sheep- and dairy-grazed pastures

Schon Nicole L. 1, Mackay A.D.

2, Minor M.A.

3

1Ecology, Institute of Natural Resources, Massey University, Private Bag 11222, Palmerston North 4442, New

Zealand

2AgResearch Grasslands, Private Bag 11008, Palmerston North 4442, New Zealand

3 Ecology, Institute of Natural Resources, Massey University, Private Bag 11222, Palmerston North 4442, New

Zealand

Anecic earthworms play an important role both the development and maintenance of soil structure

and in the incorporation and initial mixing of plant litter. A previous study, has found the distribution

of anecic earthworms patchy in New Zealand pastoral – Session 4 land, limiting their potential

contribute to ecosystem services. In this study earthworm populations were estimated as part a larger

project examining soil invertebrates in New Zealand pastures. Nine different management regimes in

sheep-grazed pastures and 17 in dairy-grazed pastures across ten locations in the North and South

Islands were investigated. Other factors which were assessed included stocking rates, fertiliser use,

soil fertility, and soil structure. Peregrine anecic earthworms (Lumbricus terrestris, Aporectodea

longa) were present in most flat and rolling, dairy-grazed pastures, with a biomass of up to 400 g wet

weight/m2. The response of anecic earthworms to increasing live-weight loading (i.e. dairy cow

stocking rates) and nutrient inputs was observed in a well-structured Allophanic soil. Anecic

earthworms tended to increase as pasture production and other inputs increased (14 to 155 g wet

weight/m2). This probably reflects both increased food resource input into the soil environment and

the lower susceptibility of anecic earthworms to stock treading pressure; with their ability to burrow

deeper to avoid physical damage from livestock hooves gives them a competitive advantage over

epigeic earthworms. At sites with poorly structured soils such a response was absent, indicating that

the physical environment was a limiting factor. The ability of anecic species to feed on surface litter

yet to escape to depth to avoid treading pressure suggests that they may offer an option for

maintaining the service of litter incorporation in the soils where stock treading limits epigeic

earthworm populations. This study confirmed, what the early study showed, that anecic earthworms

have a patchy distribution in New Zealand pastures. The absence of peregrine anecic species in

sheep-grazed hill-country pastures was notable. Some indigenous anecic species (especially

Octochaetus multiporus) were present in low fertility hill-country, but appear to play little role in the

provision of soil services once soil fertility and stocking rate are increased.

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Oral – Session 4

Manipulated earthworm populations decrease runoff and erosion rates in

arable land with distinct soil tillage treatments

Valckx Jan1, Govers Gerard

2, Hermy Martin

1, Muys Bart

1

1 Department of Earth and Environmental Sciences, Division Forest, Nature and Landscape, Katholieke

Universiteit Leuven, Celestijnenlaan 200E, BE-3001 Leuven, Belgium 2Department of Earth and Environmental Sciences, Physical and Regional Geography Research Group,

Katholieke Universiteit Leuven, Celestijnenlaan 200E, BE-3001 Leuven, Belgium

Earthworms as physical ecosystem engineers may affect key erosion-controlling soil (surface)

characteristics (e.g. soil cover and roughness, surface depression storage capacity, water infiltration

capacity, porosity) through the formation and maintenance of biogenic structures such as casts,

middens, and burrows. Although many studies observed decreased erosion rates under conservation

tillage systems as compared to conventional ploughing, and numerous others showed increased

earthworm abundance and related macroporosity under conservation tillage, the net effects of

earthworm bioturbation on soil erosion in arable land have not yet been systematically studied, at

least not at an appropriate scale. The objectives of this study were to gain insights in the relationships

between (1) soil tillage system, (2) earthworm biomass and species composition, and (3) sediment

loss through water erosion, thereby disentangling the much acclaimed net positive effects of

earthworms on soil erosion control in arable land. A large field experiment on moderately sloping

sandy loam soil in central Belgium, representative for much of the intensive crop culture in the

Western European erosion-prone loam belt, was carried out during 2005-2008. In each of three

different soil tillage treatments – shallow reduced (non-inversion) tillage (RTs) , deep reduced (non-

inversion) tillage (RTd), and conventional (inversion) tillage (CT) – earthworm populations were

either reduced by capturing using electro-shocking, increased by introducing commercially obtained

Lumbricus terrestris L., or remained unmanipulated as controls in 8 × 8 m² plots. Earthworm

fluctuations were monitored biannually. Erosion and runoff rates were measured at two scales

through rainfall simulations and experimental runoff discharges in spring 2007 and 2008, together

with relevant physical soil (surface) variables. Anecic earthworm biomass was identified as an

important independent variable in runoff and erosion regression models, next to plot slope, soil

moisture content and rainfall intensity. Erosion rates exponentially decreased as a function of anecic

earthworm biomass. Path analysis using structural equation modelling revealed that soil tillage had a

profound effect both on soil erosion and earthworm abundance, but also that anecic earthworm

biomass in itself contributed to soil erosion reduction. This study underlines the importance of

earthworms as conservators of the valuable soil resource against degradation by erosion in the

studied agro-ecosystem, and the need for promotion of appropriate soil ecosystem management by

the farmer supportive of populations of anecic earthworm species (non-inversion tillage, direct

drilling).

125


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Oral – Session 4

Nitrous oxide emission as a function of earthworm feeding strategy

Van Groenigen J.W., Paul B. , Lubbers Ingrid, Brussaard Lijbert

Wageningen University

Recent studies suggest that earthworm activity leads to increased emissions of the potent greenhouse

gas nitrous oxide (N2O). This is especially salient as, in terms of global warming potential, it may

negate the supposed capacity for earthworms to increase carbon stabilization in soil organic matter.

However, the exact pathway behind this effect is not clear. In a series of three experiments, we

studied how N2O emissions differ between earthworm species with different feeding strategies. In a

first experiment, dried grass residue (Lolium perenne) was applied on top of a loamy soil or mixed

through the soil, and N2O-emission was measured for three months. Treatments included presence of

the epigeic earthworm Lumbricus rubellus or the anecic earthworm Aporrectodea longa. Cumulative

N2O-emissions increased significantly for both species. The strongest effect was measured for L.

rubellus, where N2O-emissions significantly increased from 56 to 789 µg N2O-N kg-1 soil, but only

when residue was applied on top of the soil. In a second experiment we determined the effect of

epigeic (L. rubellus) and/or endogeic (Aporrectodea caliginosa) earthworms on N2O-emissions in the

presence of 15

N-labeled radish residue (Raphanus sativus subsp. oleiferus). Both species showed

significant increases in N2O-emissions, which differed with residue application method. The

strongest effect was measured for A. caliginosa when residue was mixed into the soil, increasing

emissions from 1350 to 2223 µg N2O-N kg-1 soil. L. rubellus only resulted in elevated N2O-

emissions when residue was applied on top. In a third experiment, we determined whether the

earthworm-induced N2O effect can be explained by the depth they can mix in the residue from the

top. In a mesocosm (50 cm depth, sandy soil) study we confined individuals of L. rubellus to the top

10 cm depth with a nylon mesh, or individuals of the anecic L. terrestris (L.) to 10, 30 or 50 cm

depth. Cumulative N2O emissions from maize residue were highest from the L. rubellus treatment at

10 cm confinement depth, but did not differ significantly with L. terrestris at 10 cm confinement

depth. Lowest emissions were found for L. terrestris confined to 30 cm. We conclude that

earthworm-induced N2O emissions differ considerably between earthworm species, which is related

to feeding strategy. Differences between epigeic and anecic earthworms can at least partly be

explained by the fact that the latter mix residue deeper into the soil.

126


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Oral – Session 4

Effect of earthworms on priming effect intensity and involved microbial

communities

Bernard Laetitia. 1, Chapuis-Lardy L.

2, Razafimbelo T.

3, Pablo A.L.

4, Legname E.

1, Brüls

T. 5, Brauman A.

1, Chotte J.L.

1, Blanchart Eric

1,

1Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, 2 Place Viala, 34060 Montpellier,

France 2Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, LEMSAT, Route des Hydrocarbures,

Bel-Air, Dakar, Senegal 3Université d'Antananarivo, Laboratoire des Radio-Isotopes, BP 3383, 101 Antananarivo, Madagascar

4Institut de Recherche pour le Développement (IRD), UMR Eco&Sols, 2 Place Viala, 34060 Montpellier,

France 5CEA, Genoscope, 2 rue Gaston Ccrémieux, 91000 Evry, France

Priming effect is defined as a stimulation of the mineralization of soil organic matter due to fresh

organic matter supply. This process is likely based on a competition between microorganisms for the

access to the ―fresh‖ carbon and is depending on the availability of soil nutrients. This poorly studied

process can have important consequences on the fate of soil organic matter and on the management

of residues or amendments in agricultural soils. The understanding of this process is thus of particular

significance in southern countries where soil fertility is essentially based on the management of

organic matter. Earthworms are ecosystem engineers known to affect the dynamics of soil organic

matter. Geophagous endogeic earthworms ingest large amounts of soil and assimilate a part of

organic matter it contains. During gut transit, microorganisms are transported to new substrates and

their activity is stimulated by (i) the production of limited amounts of readily-assimilable organic

matter (earthworm mucus), and (ii) the possible presence of fresh organic residues in ingested soil.

The objective of our study is to see (i) whether earthworms impact on the priming effect intensity

when a fresh residue is added to the soil, and (ii) whether this impact is linked to a

stimulation/inhibition of bacterial taxa. To achieve this goal, a tropical soil from Madagascar was

incubated in the laboratory, amended with a 13

C highly enriched, finely ground wheat residue, in the

presence or absence of a peregrine endogeic tropical earthworm, Pontoscolex corethrurus. Emissions

of 12

CO2 (respiration from the soil) and 13

CO2 (respiration from the residues) were followed during

24 days. The coupling between DNA-SIP (Stable Isotope Probing – separation between 12

C and 13

C

labelled bacteria) and pyrosequencing (both on 12

C and 13

C labelled bacteria) on samples with or

without earthworms was realized. Results showed that the activity of earthworms led to a stimulation

of the mineralization of wheat residues, increased priming effect intensity and modified its dynamics.

Several bacterial phylogenetic groups stimulated by earthworms were identified, some of taxa

feeding on fresh residues and others on soil organic carbon.

127


5th to 10


Oral – Session 4

Anecic earthworm removal in the field: Putting keystone species to the test

using stable isotopes

Keith Aidan M. 1, Schmidt Olaf

2, Boots Bas

2, Stromberger Mary

3

1 University College Dublin & Centre for Ecology and Hydrology, UK

2University College Dublin, Ireland

3Colorado State University, USA

One of the greatest challenges of soil biodiversity–functions research is to conduct experiments under

realistic field conditions. Previous field studies have extracted the entire earthworm community first

and then reintroduced ambient or elevated communities. The main disadvantages of this approach are

that the survival of reintroduced earthworms is unknown and that the functions cannot be ascribed to

particular species or ecological groups. The objective of this research was to test experimentally the

status of anecic earthworms as keystone species, by conducting a field experiment in which anecic

species were removed selectively, with minimum disturbance to the soil–plant system and remaining

earthworm community. Anecic earthworms at the study site (mainly Lumbricus terrestris) were

expelled from removal treatment plots (by injecting dilute mustard oil solutions into burrows) at the

start of the experiment and repeatedly thereafter for 2 years. Stable isotope tracers were then used to

quantify the effect of removal on the incorporation and decomposition of green cover crop residues

into plant functional groups and co-occurring earthworm species. Specifically, anecic functions were

assessed in terms of the fate of 13

C and 15

N from surface-applied, dual-labelled plant residue,

measured 39 days after residue application. Addition of mustard residues strongly stimulated above-

ground plant biomass production and residue-derived 15

N was taken up by grasses, herbs and, to a

lesser extent, legumes. However, removal of anecic earthworms did not affect plant biomass or N

uptake, neither did it have systematic effects on the assimilation of residue N and C by other

earthworm species. Some ecologically similar species responded differently, for instance, L. rubellus

was much more enriched in removal plots, but L. festivus was less enriched. This could reflect

reduced food competition from L. terrestris, but it could also reflect differences in migration

behaviour. N and C isotopic compositions of the 8 earthworm species studied here clearly reflected

typical endogeic (soil feeding) and anecic/epigeic (litter feeding) feeding behaviours. While selective

removal of anecic earthworms was successful, mustard residues acted as strong attractants to

earthworms in this unfertilised grassland system and removal plots were quickly recolonized by

anecic conspecifics as indicated by lower isotopic enrichment levels in removal plots.

128


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Oral – Session 4

Can earthworms help us sequester C? Production and dissolution rates of

earthworm secreted calcium carbonate

Hodson Mark. E 1

, Lambkin Denise1, Gwilliam Kerry

1, Layton Caroline

2, Canti Matt

3,

Piearce Trevor4

1Dept. Soil Science, University of Reading

2Dept. Geography, University of Reading

3English Heritage

4Lancaster Environment Centre, Lancaster University

Earthworms secrete granules of calcium carbonate. These are potentially important in the terrestrial

carbon cycle and are routinely recorded in archaeological studies of Quarternary soils.

Production rates of calcium carbonate granules by the earthworm Lumbricus terrestris L. were

determined over 27 days in a range of soils with differing chemical properties. Production rate varied

between soils, lay in the range 0 - 4.3 mgCaCO3 ·earthworm-1

·day-1

with an average rate of 0.8

mgCaCO3 ·earthworm-1

·day-1

and was significantly correlated (r = 0.83, p < 0.01) with soil pH. In a

longer term experiment earthworms repeatedly produced comparable masses of granules though over

time the earthworms lost mass and this led to a decrease in granule production. Translation of

individual earthworm granule production rates into masses of C precipitated per hectare of land per

year depends heavily on estimates of earthworm numbers. Using conservative values suggests a rate

of 18 molCaCO3 ·ha-1

·yr-1

. Upper limit estimates are in the order of 42 000 molCaCO3 ha-1

yr-1

.

Data obtained from flow-through dissolution experiments conducted in NaHCO3 solutions to buffer

the effects of atmospheric CO2 were fitted to the empirical calcite dissolution relationship r = k(1-SI)n

where r = dissolution rate, k = a rate constant, SI = saturation index and n = the reaction order. For

granules we obtained values of k = 1.72 x 10-10

and 3.51 x 10-13

mol· cm-2

·s-1

for rates normalised to

BET and geometric surface areas respectively and n = 1.8. Geometric surface area normalised

dissolution rates were similar to those reported elsewhere for both inorganic and biogenic calcite

indicating that, for calcite at least, geometric surface area is a good proxy for reactive surface area.

Leachate chemistry from leaching column experiments using soil containing 1% by mass granules

was unaffected by granule dissolution though, as assessed by changes of granule mass, dissolution

did occur. Trends in dissolution rate referenced to soil chemistry corresponded to predictions made

by the SLIM model for dissolution of limestone in soil and indicate that if Ca concentration builds up

to critical levels in soils, granule dissolution will slow or even stop and granules be preserved

indefinitely.

The standing mass of granules in soil will be a function of both production and dissolution rates and

the above data need to be combined to allow an assessment of the importance of granules in the soil

C cycle to be made.

129


5th to 10


Oral – Session 4

Earthworm ecosystem services in organic and biofuel agriculture

Wratten Stephen D.1, Sandhu Harpinder

2, Boyer Stephane

1, Orre Sofia

1

1Lincoln University, New Zealand

2CSIRO, Adelaide, Australia

Although Charles Darwin's last book emphasised the importance of earthworms in soil systems, the

importance of these soil animals has been known for hundreds of years. Organic agriculture usually

delivers a much higher level of ecosystem services than conventional farming but the economic role

of earthworms in organic systems has rarely been quantified. This presentation will include a

combination of field experimentation, value-transfer and other resource economics techniques to

explain and quantify the high value of earthworms in organic agriculture and its potential for

conventional systems. Although biofuel (ethanol) and biodiesel (crops) are increasing in area rapidly

worldwide, there is considerable concern about the effects these crops may have on the environment,

including ecosystem services such as biological control, pollination and soil processes. This work has

evaluated a range of potential biodiesel crops for yield and economic benefit. The latter includes a

resource economics approach which evaluates the external costs of biodiesel crops on soil ecosystem

services.

130


5th to 10


Oral – Session 4

Are earthworms good or bad for the soil greenhouse gas balance? A review

Lubbers Ingrid, Brussaard Lijbert, Van Groenigen J.W.


The soils of the world are an important source of all three major greenhouse gases (GHG‘s).

Terrestrial ecosystems have contributed as much as half of the anthropogenic carbon dioxide (CO2)

emission over the past two centuries; soils are by far the dominant source of nitrous oxide (N2O)

emissions; and wetlands and landfills dominate global methane (CH4) fluxes. Yet, soils can also be a

sink for these three GHG's through carbon sequestration, biological reduction of N2O, and increased

methanotrophy. The GHG balance of the soil is therefore dependant on soil management. This raises

the question to what degree soil ecosystem engineers such as earthworms can affect the soil GHG

balance. It has been suggested that earthworms can stabilize fresh organic C in soil organic matter.

Furthermore, several studies have reported effects of earthworms on emissions of N2O or CH4. Here

we review the available literature covering the effects of earthworm activity on the soil GHG

balance, and identify knowledge gaps that need to be addressed. With regard to C, many studies

suggest earthworm-induced C stabilization in soil organic matter. However, measurements were

almost exclusively indirect (measuring new C in supposedly more stable soil fractions), had a very

short duration (less than 4 months), or earthworm effects were confounded by other effects (e.g.

tillage). For N2O, a growing body of literature indicates that earthworm activity can significantly

increase N2O emissions. This was mostly measured under controlled conditions and only when a

food source was added to the system. Few studies discussed the earthworm effect on CH4 emission.

Both positive and negative effects have been reported, however, CH4 emissions appeared to be

mainly measured in landfills and contaminated soils. We conclude that available data on the effect of

earthworms on the GHG balance of the soil are fragmentary and it has certainly not been

conclusively proven that earthworms increase soil organic matter stocks. The most important

knowledge gaps appear to be (i) direct measurement of C stabilization over longer time periods; (ii)

field-based studies on N2O effects; and (iii) integral studies measuring all three effects

simultaneously. Options to fill these knowledge gaps will be discussed.

131


5th to 10


Oral – Session 4

The ecology of earthworm impact on ecosystem services

Blouin Manuel 1, Cluzeau Daniel

2, Aranda Delgado Eduardo

3, Baker Geoff

4, Brussaard

Lijbert5, Butt Kevin

R.

6, Dai Jun

7, Dendooven Luc

8, Hodson Mark

E.

9, Lavelle Patrick

10

1Université Paris-Est Créteil

2Université Rennes 1

3Terranova Lombricultores

4CSIRO Entomology

5 Wagenningen University

6 University of Central Lancashire

7South China Agricultural University

8 Instituto Politécnica Nacional

9 University of Reading

10 Institut de Recherche pour le Développement

Additional authors (in the order of appearance): -Pérès Guénola Université Rennes 1 -Tondoh Jerome

Université d‘Abobo-Adjamé -Sery Nicolas Université Paris Est -Vennetier Michel CEMAGREF -

Brun Jean-Jacques CEMAGREF Nowadays, it is recognized that biodiversity is responsible for the

delivery of many ecosystem services. Human well-being based on these services therefore relies on

biodiversity. However, these principles are not sufficient to manage biodiversity in a way that

optimizes the delivery of ecosystem services. Research on biodiversity and ecosystem services has

explored two distinct perspectives. From a side, studies on the link between diversity as a whole and

ecosystem functioning address theoretical aspects of the role of organisms, with a focus on stability,

complementarity, insurance and limited focus on the specific role played by each taxon. On the other

side, the importance of biodiversity in ecosystem services is discussed from an economical

perspective, with mapping of offer and demand and financial evaluation. The gap between these

approaches can be filled by assessing the ecological role of specific taxa in the delivery of services.

Several phenomena and practices can help evaluate the impact of earthworms on services:

conservation, (re)colonisation, inoculation and spreading of biogenic products as vermicompost. A

group of international experts has reviewed the literature on earthworm contribution to such

ecosystem services as primary production, carbon sequestration, maintenance of the soil structure,

nutrient cycling, water regulation and depollution. Documentation on these services is highly

heterogeneous: for some of them we only had indirect evidences, others are studied in dedicated

protocols. Some have been studied all around the world, others in restricted areas. Some are relevant

only in specific land uses, others everywhere. Some have been studied in the field, other in the

laboratory. For some of them, ecological mechanisms are well understood, not for others. This

review aims at helping people involved in conservation, land management or industry to have a

global knowledge on earthworms and ecosystem services, in the aim to promote a sound and efficient

use of the largest animal biomass in terrestrial ecosystems.

132


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Effect of Pontoscolex corethrurus on aggregate formation, water

infiltration and phosphatase activities over a maize-mucuna crop

Alarcón Gutiérrez Enrique1, Palma Arriola O.B.

2, Landa Guerrero Yeudiel

2, García Pérez

José Antonio2, De los Santos Bailon Martín

2, Barois Isabelle

2.

1 Instituto de Investigaciones Forestales, Universidad Veracruzana, Xalapa, Veracruz,

2 Instituto de Ecología A.C. Xalapa, Veracruz, Mexico

The aim of this study was to evaluate the effects of the earthworm Pontoscolex corethrurus in the: i)

enzymatic activity of acid and basic phosphatases from the leachate; ii) leachate pH; iii) water

infiltration; iv) leachate volume and v) soil macroaggregates, classified into two categories: 1)

biogenic A (3 cm < x), B (1 cm < x <3 cm), C (0.5 cm < x <1 cm), and 2) physicogenic D (3 cm < x),

E (1 cm < x < 3 cm), F (0.5 cm < x <1 cm). Sources of variation were: Pontoscolex corethrurus (P),

Mucuna pruriens (M) and Zea mays (Z) with their different combinations, making a total of eight

treatments: PZM, ZM, PM, M, PZ, P, Z and C (control). Data were analyzed using STATISTICA

8.0, using nonparametric analysis (Kruskal-Wallis). Results indicated significant differences (p

<0.05) in infiltration between P (0.03 cm3s

-1) and Z (0.002 cm

3s

-1) treatments at 30 days, where P was

the highest. At 60 days, differences were observed between treatment C (0.053 cm3s

-1) and PZ

(0.0011 cm3s

-1). The volume of leachate was higher in M (2.30 L) treatment. In regards to leachate

pH, there was significant difference (p <0.05) at 60 days between M (pH 7.74) and PZM (pH 6.69)

treatments. On the other hand, at 120 days, basic phosphatase activity was higher in the P (0.682

nkat/mL) treatment and acid phosphatase activity was higher in the PZ (6.79 nkat/mL) treatment.

Finally, macroaggregates type C (0.5 cm < x <1 cm) showed significant difference (p <0.05) between

PZM (108.61 cm2) and M (0.0 cm

2) treatments; this possibly due to the formation of aggregates in

fine roots (PZM), and the lack of earthworms (C). The earthworm P. corethrurus promotes

infiltration by a short period of time and may promote enzyme activity in the leachate and the

formation of aggregates associated with fine roots.

133


5th to 10



The role of New Zealand endemic earthworms in mine restoration


Bio-Protection Research Centre, Lincoln University

Among the soil-dwelling species that are likely to be important early in mine restoration, earthworms

are particularly good candidates. They provide ecosystem services that are likely to facilitate and

accelerate the restoration of a functional ecosystem. These services include increasing topsoil

fertility, providing food resources for a wide range of predators and the recycling of waste organic

materials. Despite their ecological importance, earthworms have been poorly considered in mine

restoration. Most restoration plans focus only on vegetation and above-ground macro-fauna, while

the aim should be to restore functioning ecosystems above and below ground. With the aim of

developing recommendations for better management of earthworm communities after opencast

mining, we evaluated the response of an endemic earthworm community to mining activities and

rehabilitation treatments currently used in an opencast coal mine in New Zealand. The main

treatments were vegetation direct transfer (VDT) and vegetation replanting in soil that has previously

been stockpiled. VDT consists of removing pieces of land, including the vegetation and the topsoil

and transferring them to another area where the land ‗jigsaw‘ is reconstituted. The present work

showed that this method was efficient in conserving earthworm communities, both in terms of

biomass and species diversity. Soil that has been stored in high stockpiles, on the other hand, was

mostly anaerobic and contained very few earthworms. When re-spread and replanted, such soil

contained a depauperate earthworm community and required enhancement of its fauna to restore its

ecosystem functions. Stockpiling, re-spreading and replanting is a common practice in opencast mine

rehabilitation. However, based on our study it does not provide a satisfactory outcome for earthworm

communities and is likely to be detrimental for most soil invertebrates as well as the ecological

communities that depend on them.

134


5th to 10



Can reduced tillage and field margins improve soil water dynamics

through stimulation of earthworms?

Crittenden P.S.J. 1, Pulleman Mirjam

1, Brussaard Lijbert

1, Heinen M.

2

1 Department of Soil Quality, Wageningen University

2 S Alterra

Concern is growing among Dutch farmers about water logging and soil compaction given that

climate change scenarios for The Netherlands predict exacerbated extreme rainfall events and

drought periods (MNP, 2005; KNMI, 2006). Conservation tillage practices, such as non-inversion

tillage, are being implemented to reduce compaction, improve the infiltration and water storage

capacity of soils, and stimulate biological activity. The ability of the soil to absorb and retain water is

considered an important ecosystem function. After initiating reduced tillage practises there may be an

increase in earthworm populations and diversity due to reduced disturbance, but this process will

depend on their presence in the arable field and near-by landscape elements, and on their dispersal

rates (Hoogerkaamp et al., 1983; Mather and Christensen, 1988). These non-productive landscape

elements (e.g. field margins) are managed to promote above-ground farmland biodiversity but can

also have an impact on soil macrofauna (Smith et al., 2008). This project is investigating the

complementarity of soil and landscape management strategies for the enhancement of ecosystem

services. A replicated field experiment was initiated in 2009 to investigate the relationships of non-

inversion tillage and earthworm populations and diversity with water infiltration and retention under

conventional and organic farming practices. A second trial investigates earthworm populations in

field margins and distributions in adjacent arable land. Earthworm species diversity and soil physical

property data will be presented from two field seasons. References: -Hoogerkamp, M., Rogaar, H.,

Eijsackers, H.J.P., 1983. Effect of earthworms on grassland on recently reclaimed polder soils in the

Netherlands. In: Satchell, J.E. (Ed.), Earthworm Ecology. Chapman and Hall, London, pp. 85-105.

- KNMI, 2006. Klimaat in de 21e eeuw, vier scenario‘s voor Nederland. In: De Bilt. -Mather, J.,

Christensen, O., 1988. Surface movements of earthworms in agricultural land. Pedobiologia 32, 399-

405. - MNP, 2005. Effecten van klimaatverandering in Nederland. In: Milieu- en Natuurplanbureau. -

Bilthoven Smith, J., Potts, S.G. Woodcock, B.A., Eggleton, P., 2008. Can arable field margins be

managed to enhance their biodiversity, conservation and functional value for soil macrofauna?

Journal of Applied Ecology 45, 269-278.

135


5th to 10



Biological Alternatives to the use of pesticides in banana

plantations of Colombia

Chagüeza Yamileth

1, Asakawa Neuza

2, Bolaños Martha

3, Lavelle Patrick

4,

Velasquez Elena1

1Universidad Nacional de Colombia, sede Palmira, Colombia

2Centro Internacional de Agricultura Tropical, Cali, Colombia

3CORPOICA, Palmira, Colombia

4UMR BIOEMCO, Université Pierre et Marie Curie/Institut de Recherche pour le Développement, Paris,

France

Productivity and sustainability in Banana plantations is currently threatened by a large number of

pests and diseases. The continuous use of nematicides and other pesticides is only solving the

problem in short term and creates serious problems of human and ecosystem health. Our project

evaluates the manipulation of different biological components as alternatives to the use of pesticides.

We experimented the effect on phytoparasitic nematodes of 4 different treatments: 1. application of

lixiviates from banana trunks; 2. application of lombricompost; 3. Inoculation of mycorhizae in roots

and 4. application of the FBO (Fertilization Bio Organique) patented technique that creates islands of

high fertility with application of low (Gliricidia branches) and high quality (lombricompost) organic

materials in trenches inoculated with local endogeic earthworms. Before the experiment, we assessed

soil aggregation (Velasquez et al., 2007 method) and population of mycorhizae, saprophytic and

plant parasitic nematodes and we found a significant relationship among i) aggregates of biological

origin (earthworm casts) and mycorhizal spores; ii) Endogeic earthworms and non aggregated soil;

iii) polyhumic earthworms and organic matter content and iv) nematodes with root and physical

aggregates. Treatments were applied to 93 plants. Six months after, we measured mycorhizal

colonization frequency, the occurence of arbuscules in the whole root system, the number of

mycorhizal spores in 100g of soil and densities of phytoparasitic and saprophytic nematodes in roots

and soil. PCA analysis on soil communities of the 31 plants that survived an unexpected drought

showed 1) very low numbers of all nematode groups and mycorhizae in the control treatment as

opposed to our biological treatments, with FBO having the highest numbers of organisms and the

lombricompost and mycorhizae treatments with intermediate values (axis 1 of the PCA analysis); 2)

relatively larger numbers of mycorhizae and saprophytic nematodes in the FBO treatment as opposed

to other biological treatments.

136


5th to 10



Earthworms in the Quesungual agroforestry system and their role in

aggregate-associated soil organic matter and nutrient dynamics.

Fonte Steven1,2

, Barrios Edmundo2,3

, Six Johan1

1 Department of Plant Sciences, University of California, Davis, California, USA

2 CIAT-TSBF-LAC Institute, Cali, Colombia

3 World Agroforestry Centre, Nairobi, Kenya

The Quesungual agroforestry system from the mountains of western Honduras has been promoted as

a viable alternative to traditional slash-and-burn agriculture in tropical dry forest regions of the

Americas. Although mulch application and the lack of burning in this system can have notable

impacts on soil macrofauna, the influence of Quesungual management on earthworm populations has

not been fully quantified. Even less is understood about the role earthworms in regulating soil

organic matter (SOM) and nutrient dynamics in the Quesungual system. We present a summary of 3

field studies conducted in Honduras aimed at 1) quantifying the influence of the Quesungual

management on earthworm abundance and diversity and 2) understanding how earthworms in this

system interact with agroecosystem management to influence soil aggregation, SOM and nutrient

cycling. The first study examined earthworm populations and soil properties within a replicated field

trial comparing the Quesungual system with traditional slash-and-burn agriculture and undisturbed

secondary forest. Quesungual management significantly increased earthworm populations relative to

slash-and-burn agriculture (with intermediate values for secondary forest). A second experiment

manipulated earthworms (Pontoscolex corethrurus) using microcosms within the replicated field trial

mentioned above. Earthworms were found to reduce total soil C in the surface soil across all

management systems and to facilitate the loss of fertilizer N within both the Quesungual and slash-

and-burn plots. A third study employed field mesocosms (each containing a growing maize plant) to

manipulate earthworms (P. corethrurus) and residue additions at a nearby study site. In the presence

of active plant roots, earthworms improved aggregation and C storage within stable aggregate

fractions, but only when residues were added to the soil surface. Earthworms also increased the

uptake of fertilizer N by maize plants and reduced total and available P across all residue treatments.

Considered together these studies suggest that earthworms are promoted by Quesungual

management, but that their beneficial impacts on SOM and nutrient cycling depend on the proper

management of plants and the addition of sufficient residue inputs.

137


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Earthworm-induced N2O emissions in a sandy soil with surface residue

application

Giannopoulos Georgios2, Van Groenigen J.W.

1, Pulleman Mirjam

1

1 Department of Soil Quality, Wageningen University, PO Box 47, 6700 AA Wageningen, The Netherlands.

2 School of Biological Sciences, University of East Anglia, NR4 7TJ, Norwich, United Kingdom.

Earlier research with endogeic and epigeic earthworm species in loamy soils has shown that both

earthworm groups can increase N2O emissions when crop residues are incorporated into the soil by

epigeic earthworms. This effect was related to changes in soil organic matter (SOM) distribution in a

loamy soil, characterized by a well-aggregated stable soil structure. The current study focused on

sandy soil which typically contains less SOM and stable soil aggregates. We aimed to i) quantify

N2O emissions in sandy soil as affected by endogeic and/or epigeic earthworm species and ii) relate

changes in N2O emissions to earthworm-induced changes in OM distribution and physical soil

fractions. A 90 day 15

N-tracer mesocosm study was conducted using sandy soil, with the endogeic

earthworm species Aporrectodea caliginosa (Savigny), the epigeic species Lumbricus rubellus

(Hoffmeister) and both species combined. 15

N-labeled radish (Raphanus sativus cv. Adagio L.)

residue was placed on top the soil. An additional treatment was included with residues manually

incorporated into the soil and no earthworms. L. rubellus significantly increased N2O emissions from

228 to 859 μg N2O-N kg-1

. A. Caliginosa did not significantly affect N2O emissions and no

significant interactions between earthworms species were observed. When residues were manually

incorporated, N2O emissions were significantly (p<0.008) lower (509 μg N2O-N kg-1

) compared to

incorporation by L. rubellus. The amount of C and N in the size fraction >250 μm was significantly

increased by the presence of L. rubellus and/or A. Caliginosa, but the 15

N enrichment of that same

fraction was only increased in the presence of L. rubellus. We conclude that earthworm-induced N2O

emissions in a sandy soil with surface residue application are affected by L. rubellus, but not by A.

Caliginosa, reflecting the feeding strategies of the different ecological groups. In contrast to earlier

studies in loamy soil, no interactive effects of the two species on N2O emissions were found. The

higher N2O emissions with L. rubellus compared to manual mixing, could be due to earthworm-

induced changes in OM distribution or microbial processes in the soil.

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Impact of the earthworm Pontoscolex corethrurus on the porosity of the

soil, observations in a pot experiment by CT scanning and standard

methods

Gomez-Tagle Alberto (Jr.)1, Morales Chávez Rafael

1, Alarcón Gutiérrez Enrique

2, Stoopen

Rometti Miguel2, Zarate Reyna Paula

4, Barois Isabelle

4

1 INIRENA, Universidad Michoacana de San Nicolás de Hidalgo, Morelia Mexico

2 Instituto de Investigaciones Forestales, Universidad Veracruzana, Xalapa, Mexico

3 CT Scanner de Mexico, D.F. Mexico

4 Instituto de Ecología A. C., Xalapa, Mexico

Earthworms as ecosystem engineers have an important impact on the soil porosity and the water flux;

although standard methods of soil porosity do not allow to observe clearly this impact. In order to

study in a detailed form this effect a pot experiment was set. Treatments included: 1) P. corethrurus

with maize and Mucuna (PZM), 2) P. corethrurus with Mucna (PM) 3), P. corethrurus with maize

(PZ) and 4) the control which is the soil alone (T). The Mucuna was put as crushed litter on the

surface at the beginning of the experiment. The soil is repacked disturbed luvisol from 0-10cm

already ground to 5 mm and dumped into 25 cm lower diameter, 35 cm higher diameter and 40 cm

height truncated cone shaped plastic pots. In treatments PZM, PM and PZ and 10 adult earthworms

were introduced in the pot at the beginning of the experiment. The experiment was conducted for a

whole growing season for maize. At the end of it, CT Scan images were acquired with a 64 multislice

helicoidal CT Scanner (GE. BCT light speed). Macroporosity 3D reconstruction and analysis was

conducted using a digital crop monolith of 15.0x15.0x10.0 cm from each treatment to avoid

unnatural preferential burrow paths at the soil-pot contact. Image analysis was done by means of

AMIRA 4.x software (Visage Imaging). After scanning, soil cores were extracted with a cylinder (5.0

cm diameter x 5 cm height) to calculate Bulk Density at two depths (0-10 cm and 20-30 cm) in each

pot and from it obtain total porosity. Results indicate that 3D images of treatments with earthworms

(PZM, PM, PZ) present a complex and large macro porosity network. Treatments with earthworms

developed 6,162.85 to 7,468.43 mm3 of macropore space (2.36x10

-1 to 3.32x10

-1 %) while treatments

without earthworms developed only 7.5-9.9 mm3 of macropore space (3.33x10

-4 to 3.98x10

-4 %). The

difference in soil macropore volume between treatments with earthworm and those without

earthworms is over 700 times The core technique did not evidence any differences in total porosity

among the treatments . The porosity range was between 48 and 49 %. These results suggest that the

core method may have few sensitivity to significant changes in macropore space. As mentioned

earlier in literature, our analysis allowed us to corroborate that earthworms had a dramatic impact

(two orders of magnitude) in soil macropore space and structure. CT Scan image processing and 3D

image reconstruction of macropore soil space is a powerful tool in the field of earthworm ecology

and soil bioengineering. This work was partly supported by the project Conservation and Sustainable

Management of Below Ground Biodiversity financed by GEF/UNEP and executed by CIAT/TSBF.

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Field assessment of burrow reuse by Lumbricus terrestris L.

Grigoropoulou Niki, Butt Kevin R. University of Central Lancashire, School of Built and Natural Environment, Preston PR1 2HE, UK

A short term field experiment was set out to investigate evidence of burrow inheritance or re-use by

Lumbricus terrestris. This was established in a temperate broadleaved woodland and involved

removal of L. terrestris from a number of 1 m2 plots with subsequent monitoring of colonisation in

these areas. Manipulation of L. terrestris number in plots resulted in 4 treatments. 1. Removal of L.

terrestris while preserving their burrows (Rem) from (a) 1 m2 open plots (RemO) to examine re-use

and inheritance of vacated burrows by other conspecifics and offspring (b) 1 m2 enclosed plots

(RemE) to examine inheritance of vacated burrows by offspring. 2. Removal of L. terrestris and

cocoons through soil sterilisation and the destruction of burrow systems (Ster) from (a) 1 m2 sterilised

open plots (SterO) to examine colonisation of empty space (b) 1 m2 sterilised enclosed plots (SterE)

used as a control for the sterilisation method. Each of the treatments was replicated 3 times resulting

in a total of 12 plots. Enclosure was achieved by construction of a plastic fence which extended 0.3 m

below and 0.3 m above the soil surface. The cumulative mean percentage of vacated burrows being

re-used by conspecifics was compared in plots of all treatments on a monthly basis over a period of

10 months. A significantly (p<0.01) greater proportion of vacated burrows were re-used in RemO

plots (mean±S.E. 50.27%±3.57%) compared with RemE plots (38.48%±3.49% m-2

). A similar

number of L. terrestris middens was observed in plots of SterE treatment (mean±S.E. 29.33±2.33)

and SterO treatments (23.57±1.28 m-2

), over the entire duration of the experiment. The spatial

distribution of L. terrestris middens was examined using Ripley‘s K function and midden

permanence using the bivariate Ripley‘s K function. Point patterns were found to be regularly

distributed at mean(±S.E.) distances ranging from 0.084±0.029 m to 0.130±0.063 m in 43.27%

(n=45) of instances. Patterns of vacated and reused burrows in Rem treatment were found to be

aggregated in all six plots at distances ranging from 0.022±0.015 m to 0.132±0.043 m. These

observations suggest that L. terrestris burrows could be recycled or inherited. Such behaviour could

be a means of minimising energy expenditure for burrow construction. Results from this experiment

are significant as they provide evidence of burrow reuse by conspecifics and further support the

views of other researchers, on possible niche inheritance in L. terrestris.

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Earthworms and soil ecosystem services at homegardens of Tabasco

Mexico

Huerta Esperanza, Van der Walls Hans, Jarquin Aaron, Ponce Alejandro, Javier Sigrid,

Magaña Mauricio

El Colegio de la Frontera Sur, Unidad Villahermosa

Home gardens have been defined as agroforestry land-use systems with multipurpose trees and

shrubs, under the management of family labour (Fernándes and Nair 1986). The main questions

addressed in this study were: plant diversity or vegetation structure in home gardens may enhance

soil earthworms diversity and abundance? Earthworms diversity and abundance may enhance soil

ecosystem services? At Tabasco, southeast Mexico we measured the diversity and abundance of

earthworms at 50 homegardens of the 5 physiographic regions of the State. 5 monoliths of

25x25x30cm in a random designed per homegarden were made, where we measured earthworms

abundance, biomass, diversity and soil physicochemical properties. We found significant

correlations (Pearsons and Spearman rho) between earthworms and infiltration (R: 0.3 p<0.05),

earthworms and soil organic matter (r2: 0.6 p<0.05), and earthworms and total nitrogen (r2:0.7

p<0,05). We observed a significant effect of precipitations and clay content on the presence of

earthworms. The Mountain region was the region with the highest abundance of endogeics (118.4

ind.m-2

), represented mainly by the Megascolecidae individuals (66.9 ind.m-2

). We observed that

total basal area of plants is the main characteristic that influences earthworms abundance.

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Effect of Pontoscolex corethrurus, Zea mays and Mucuna pruriens on the

respiration rate of soil

Landa Guerrero Yeudiel1, Alarcón Gutiérrez Enrique

1, Torres Jiménez Maria Guadalupe

2,

Barois Isabelle3

1 Instituto de Investigaciones Forestales/Universidad Veracruzana 2 Red de Ecología Funcional

3Instituto de Ecología, A.C

Soils are involved in the provision of many important environmental services to maintain the

ecosystems and human societies (Velasquez, 2004). Changes in land use are known as contributors to

increase atmospheric CO2 levels (Wisniewski et al., 1993), and may also have an influence on

earthworms, which play an important role in regulating processes to be held on the ground (Liu and

Zou. 2002), they are also part of the functional groups of the soil biota that contribute to the

regulation of vital processes to the functioning of ecosystems. Soil respiration is one of the most

common method to infer the level of biological activity including soil microorganisms, roots and soil

fauna. The presence of exotic earthworms significantly accelerates the decomposition of organic

matter and soil respiration, which increase the release of CO2 from soils to the atmosphere (Liu and

Zou 2002) and the incorporation of organic matter increases the respiration rate. In this study, the aim

was to evaluate the effect of Pontoscolex corethrurus, Mucuna pruriens var. utilis and Zea mays on

the respiration rate of a ferric Luvisol soil type, cultivated with maize under green house conditions,

using a multifactorial design at two levels (presence-absence), with eight treatments and three

replications each: T (Control), PZM (P. corethrurus + Z . mays + M. pruriens); ZM (Z. mays + M.

pruriens ); PM (P. corethrurus + M. pruriens); M (M. pruriens); PZ (P. corethrurus + Z. mays); Z (Z.

mays) and P (P. corethrurus). Respiration rate was monthly monitored using an environmental gas

monitor (PP Systems). Results indicate that the respiration rate of treatments Z, T and P had average

values of 0.66, 0.41 and 0.3 g CO2 ·h-1

·m-2

, respectively, corresponding to those treatments with a

relatively low respiration rate. In contrast, average values of treatments PZM, ZM, PZ, PM and M

were: 1.5, 1.1, 1.0, 0.76 and 0. g CO2 ·h-1

·m-2

, respectively, corresponding to those which had an

increased in respiration rate during the experiment. Combined treatments PZM, ZM and PZ

positively affect respiration rate over time compared with control; there was a combined effect

between organic matter, earthworms and plant on the modification of the respiration rates.

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Control of manure odorous compounds by adding Lactobacillus and

earthworm meal into piglet diet

Li Wenlii1,2

, Zeng Zhengqing1,3

, Lu Zhenyu1, Jiang Zhonghe

1, Sun Zhenjun

1

1 College of Resource and Environment Science, CAU, Beijing 100193, China;

2 College of Animal Science, Qingdao Agricultural University, Qingdao, 265200, China)

3 Feedstuff Section, Agribusiness & Food, New Hope Group, Chengdo, 610041,China)

Many effective microorganisms lived in earthworm gut have strong interaction for functioning

digestion and other physiological activities of earthworm. If some microorganisms including

lactobacillus spp. in vermicompost can be used to other animal waste management? It is little

reported. In this experiment, we added lactobacillus and earthworm meal into piglets‘ diet to

investigate their effect of on the pig performance and odorous compounds in feces. 144 piglets

(Landrace X Duroc) pigs were randomly divided into four groups, and fed with Corn Soybean Meal

diet (CS), CS+2×109cfu, CS+0.45% and CS+2×10

9cfu+0.45%earthworm respectively; Piglet‘s

performance, feces pH, the counts of E. coli, Lactobacillus, Clostridium, Eubacteria, ammonia

emission, ammonium, total nitrogen and main odorous compounds in the feces were determined. It

was turned out that when the diets was supplemented with lactobacillus, feces pH, E.coli counts, the

concentration of valeric acid, p-cresol in the feces were reduced greatly（ P<0.05） , while the

concentration of acetic acid, propionic acid in feces increased significantly（ P<0.05） . Dry material

of feces, feces pH and the concentration of indole were decreased greatly（ P<0.05） , whereas the

concentration of 3-methylindole in the feces was increased（ P<0.05） , when the piglets fed with diets

containing earthworm; the concentration of p-cresol, indole in the feces also were

decreased（ P<0.05） , increase of ammonia emission from slurry and no significant effect on feces

pH, volatile fatty acids and 3-methylindole in the feces （ P>0.05） were observed when piglets fed

with diets containing with both Lactobacillus and earthworm.

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Spatial earthworm casting activity in connection with the structure of the

vegetal cover in a tropical rainforest of the Guyana Shield

Nahmani Johanne1, Schimann Heidy

2, Blanc Lilian

2, Hättenschwiler Stephan

3,

Lapied Emmanuel4

1Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes (LIEBE), UFR Sciences

Fondamentales et Appliquées. Université Paul Verlaine, Campus Bridoux, Rue du Général Delestraint, 57070

Metz, France 2UMR Ecologie des Forêts de Guyane (EcoFoG). BP709, 97387 Kourou cedex, French Guiana, France

3Centre d'Ecologie Fonctionnelle et Evolutive (CEFE-CNRS) - UMR 5175. Département Fonctionnement des

Écosystèmes. 1919 Route de Mende, F34293 Montpellier cedex 5, France 4Bioforsk, Soil and Environment, Fredrik A. Dahls vei 20, N-1432 Ås, Norway

In a primary rainforest of the Guyana Shield (French Guiana), the earthworm surface casting activity

was quantified and mapped in connection with the distribution and size of a range of vegetal cover

types. A 50 plots regular quadrat was settled within a one hectare sloping parcel (BRIDGE project) at

the Paracou research site. Each plot corresponded to a 4m diameter circle in which number, volume

and position (according to various structure of vegetal cover) were measured. Results showed that, at

the scale of a one hectare gentle slope, surface earthworm casts were not homogeneously distributed

but significantly concentrated in middle slope, thus reaching 2.55 casts.m-2 and 2.7 dm-3. In

addition, the density of trees (of all diameter classes) and seedlings (dicotyledons), palms

(monocotyledons), dead trunks or branches, lianas on soil, emerging roots or herbaceous did not

influence the density of earthworm surface casts. However, within areas of high density of casts,

these latest were significantly more localized at the foot of trees and seedlings than other vegetal

structure. Earthworm casts were also significantly more localized at the foot or directly under hard

vegetal structures than within the litter.

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Settlement and spread of an introduced earthworm (Lumbricus terrestris

L.) population in relation to field management in boreal clay soil

Nuutinen Visa1, Butt Kevin R.

2

1MTT, Plant Production, Soils, FI-31600 Jokioinen, Finland

2UCLan, School of Built and Natural Environment, Preston PR1 2HE, UK

To study the feasibility of earthworm introduction in increasing the macroporosity and permeability

of arable clay, the deep burrowing earthworm Lumbricus terrestris L. was inoculated into an

experimental leaching field in Jokioinen, S-W Finland in autumn 1996. The species was initially

absent from the field and its immediate surroundings. The soil of the sloping (2%), sub-drained field

is Vertic Cambisol with clay content ranging from 61% in topsoil to 90% in subsoil. The field

consists of four adjacent plots (33 m x 140 m) surrounded and separated by permanent grass strips.

Inoculation took place inside the plots and in the grass strips, close to the upslope end of the field

using Earthworm Inoculation Unit technique. Mustard extraction of L. terrestris was used to follow

population trends. The first follow-up (1998) showed that the population had persisted in low

numbers, but only in the grass strips outside of the cultivated area. At second monitoring (2003),

median density was 9 ind. m-2

in the grass strips where there was evidence for dispersal to 8 m from

the inoculation points. This time a few L. terrestris were also found within the inoculation area in the

field but the median density was only 1 ind. m-2

. The third monitoring was in autumn 2009 until

which two of the plots had remained unploughed (from 2003: either grass-covered or direct drilled).

The other two plots had been ploughed the previous autumn but otherwise had similar management.

In the grass strips, population density had increased and the mean density peaked at 36 ind. m-2

close

to the points of inoculation, gradually declining down-slope with mean density of 6 ind m-2

at 55 m

distance from inoculation. Inside direct drilled plots, L. terrestris were abundantly found, with a

similar trend, from 21 ind. m-2

close to the inoculation to 2 ind. m-2

at 50 m distance. In ploughed

plots the numbers were much lower, with maximum density of 3 ind. m-2

upslope and no individuals

found beyond 34 m from inoculation. Inside the field the densities were consistently higher above the

sub-drains than between them (overall mean 6 vs. 2 ind. m-2

, respectively). It seems that the eventual

establishment of a population inside the field depended on the preceding population growth at the

plot margins which acted as sources of dispersers into the field. Low physical disturbance together

with sub-drainage were factors favouring settlement and spread of the introduced population.

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Population densities for the growth and cocoon production of the

earthworm Pontoscolex corethrurus (Annelida: Oligochaeta,

Glossoscolecidae)

Pascacio Velázquez José M., Sánchez Velásquez Lazaro Rafael., Ortiz Ceballos Angel I.

Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana

In spite of the potential of the earthworms to conserve the fertility of the soils and to increase the

agricultural and forest production, biotechnologies for the massive production, of earthworms do not

exist. In the present study was evaluated the effect of the population density of the exotic earthworm

Pontoscolex corethrurus in the cocoon production. In conditions of greenhouse, during a period of

135 days, the earthworm culture under three population densities, high, medium and low (340, 227

and 114 worm m-2, respectively) each with three replicates. Every 15 and 30 days was registered the

number and biomass of the produced cocoon and the growth of progenitor earthworms, respectively.

The results this study suggested that until the 90 days of established the experiment of the three

densities of P. corethrurus maintained a constant density and increased significantly their biomass;

nevertheless, at the end of the study (135 days) the densities were similar to the initial population

being 58.73%. The total cocoon production (71.8) was not significant between the three densities and

the major cocoon production happened to the 60 days. The preliminary conclusions suggest that the

cocoon production is determined by the environmental conditions and the greater number of cocoons

for each earthworm occurs in a low population density.

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Modelling earthworm populations and traits of earthworm activities under

contrasted soil tillage management

Piron Denis1, Cannavacciuolo Mario2, Pérès Guénola1, Cluzeau Daniel1

1UMR EcoBio CNRS_Univ-Rennes1 (France)

2ESA Angers (France)

Tillage practices greatly affect soil physical characteristics and substitute natural soil aggregation

(pedoclimatic and biological) by soil fragmentation. In other hand, conservation tillage is favourable

to soil biological activities and notably earthworm populations. An organic management system

tends to increase the density of earthworm community due to organic fertilization (as trophic

resources). If reduce tillage can be practiced in organic farming management, we can considered that

this type of management is favourable to biological activities notably to earthworm biological

activities (and thus earthworm activities (macroporosity and aggregation)), but a few work confirms

this hypothesis. In this work, two tillage managements are compared using a completely randomised

block design with three replicates (6 experimental plots): (1) traditional mouldboard ploughing

(tilling to a depth of 30 cm), (2) reduced tillage with tined tools (15 cm depth) We studied the

structure of earthworm functional groups (according to ecological groups), and quantified biological

soil pattern (generated by earthworms). So we developed a model to predict earthworm population by

superficies of soil structure patterns according to Partial Least Squares regression.

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Soil water movement is affected by two anecic species (Lumbricus terrestris

and Aporrectodea giardi)

Piron Denis1, Pérès Guénola

1, Ablain Frédérique

1, Hallaire Vincent

2, Boubrit Sarah

1,

Cluzeau Daniel1

1UMR EcoBio CNRS Univ-Rennes1 (France)

2UMR SAS INRA_Agrocampus Ouest (France)

Earthworms modify soil physical properties and thus affect soil hydraulic conductivity. Anecic group

describes large earthworm species that built some semi-vertical burrows. So, anecic burrows are

widely recognized to facilitate soil infiltration. However, Jégou et al. (2001) have shown some

pronounced differences of burrow systems (length and connection) for the two species Lumbricus

terrestris L. and Aporrectodea giardi. The aim of this study was to assess the water movement

according to these two anecic species. So, artificially packed soil columns were inoculated with two

individuals of Lumbricus terrestris (LT) or Aporrectodea giardi (AG). During 12weeks, we have

weekly measured the percolation and more, we have study the kinetic of percolation after a rainfall

simulation. Compared to soil columns that were no inoculated, our results indicate that earthworm

species have improved the water movement in soil columns. Similar percolated volumes were

measured with the two earthworm species, these volumes being twice these measured under control

columns. After a rainfall simulation, the percolation begins more late in the columns of control, only

1,5 % of water have percolated during the first hour. Respectively with LT and NG, 61,2 % and 57,8

% of water have percolated during the same time. So, the kinetics of percolation differs slightly

between the both earthworm treatments during the first hour. In LT, we observed a phase of very fast

percolation (< 5min) and then residual flows. In AG, two phases of percolation were observed: very

fast (< 5min) and then a steady percolation during 55min. In conclusion, the contrasted burrow

systems constructed by anecic does not affect significantly the percolation ratio but the rapidity of the

water movement in soil column.

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Earthworm communities and their role in the first steps of soil structure

formation in carbonate-rich alluvial soils

Salomé Clémence1, Guenat C.

2, Bullinger-Weber G.

3, Gobat J.M.

4, Le Bayon Claire

4

1University of Neuchâtel

2Ecole polytechnique fédérale Lausanne

3University of Lausanne

4University of Neuchâtel

Alluvial ecosystems are good models to understand the establishment of earthworm populations.

These ecosystems exhibit a mosaic of habitats containing strong different pedologic characteristics as

soil texture, profile depth, carbonate and organic matter contents, humus forms and vegetation cover.

Furthermore, the age of soils is very young in such fluvial systems (from a couple of years to around

a hundred years); hence, soil structure is not widespread developed and pedogenetic processes are

continuously in progress. So, the various roles of earthworms as ecosystem engineers in these

ecosystems are very interesting to investigate because of the pioneer nature of soils. In this context,

we proposed to study earthworm populations and their roles in the first steps of soil structure

formation. We hypothesise that it may vary according to both altitude and elevation from the river

bed. Thus, our three main study sites were located in Switzerland at three altitudinal levels

(subalpine, mountain and hill level). Perpendicular to this altitudinal gradient, a vegetation gradient

was also chosen reflecting the duration of soil stabilisation. Sampling was performed in carbonate-

rich alluvial soils. A total of 45 soil profiles were studied for their structure (porosity, aggregate

stability and mean weight diameter of aggregates) and their physico-chemical characteristics in the

30 first centimetres. For each soil profile, earthworms were collected twice a year; they were

counted, weighted and identified to species level. First results revealed a biomass ranging from 1g m-

2 to 160g m-2, with strong differences between and within altitudinal levels. A total of 27 species

and sub-species were recorded along the two gradients. In addition, strong differences were found

comparing ecological categories: for example, no anecic species were found at the subalpine level.

Statistical treatments of the data are still on-going to i) characterize the specific habitats of

earthworm species performing multivariate analyses (clustering and redundancy analysis) that

compile soil characteristics, vegetation units and altitudinal levels, and ii) to point out the role of

earthworms as ecosystem engineers. For this latter part, our results reveal that anecic and endogeic

species in less extend are determinant in the development of soil structure through their burrowing

and casting activities and their biomass is highly correlated with the thickness of the structured soil

layers.

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Biomass energy crop impacts on earthworms and associated cast microbial

activity

Scullion John, Ulyett James, Farrar Kerrie, Donnison Iain

Aberystwyth University

Biomass energy crops have the potential to contribute to sustainable energy targets. In the UK,

consideration is being given to their widespread planting. However, some of the crops are exotic so it

is important to consider potential ecological impacts. Litter from established field trials growing

various Salix and Miscanthus hybrids was collected and fed to Lumbricus terrestris in mesocosm

trials with and without a pre-decomposition phase. Earthworms were monitored for health and

growth, litter consumption rates and surface casting. Casts were further analysed for C, respiration

and microbial biomass. Decomposition and litter type (including different Miscanthus hybrids)

affected earthworms differently. There were also clear differences in cast microbial indices which

were not explained solely on the basis of C contents. Results suggest that at least some of the

favoured biomass energy crops may significantly alter below ground ecology and C cycling.

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Feeding ecology of Lumbricidae: the other species

Szlavecz Katalin1, Zicsi Andras

2, Csuzdi Csaba

3

1 Johns Hopkins University

2 Systematic Zoology Research Group of Hungarian Academy of Sciences

3Systematic Zoology Research Group of Hungarian Academy of Sciences

Most of our knowledge on food selection and consumption by earthworms is based upon studies

carried out with a small number earthworm species. These species are usually synanthropic,

cosmopolitan species. Moreover, soil food webs are often considered to be highly redundant, when

one species in the same functional group can replace another without altering ecological functions.

Here we compare the feeding ecology of a total of 31 lumbricid species. Most of the lesser known

species are endemic in Central Europe where many of them dominate temperate deciduous forests.

Five large anecic species [Lumbricus polyphemus (Fitzinger, 1833), L. terrestris L. 1758, Fitzingeria

platyura montana (Cernosvitov, 1932), F. platyura depressa (Rosa, 1893), F. platyura platyura

(Fitzinger, 1833) and Dendrobaena clujensis Pop, 1938] were kept in 150 cm tall laboratory

monoliths and fed hornbeam (Carpinus betulus) leaf litter. Leaf consumption rates and cast

production varied between 16 and 37 mgDM gFM-1 day-1 and 314 and 30 mgDM gFM-1 day-1,

respectively. In most cases at least 75% of the cast was deposited in the soil, but the distribution of

casts with depth varied with species. Smaller litter feeding species were kept in 50 cm tall

mesocosms and included the peregrine Lumbricus. rubellus Hoffmeister, 1843, L. castaneus

(Savigny, 1826), Dendrodrilus rubidus (Savigny, 1826), Dendrobaena octaedra (Savigy, 1826), and

the Eastern–Alpine D. auriculata (Rosa, 1897). Here both consumption and cast production rates

were smaller: 17-24 mgDM gFM-1

day-1and 2-8 mgDM gFM-1

day-1

, respectively. Endogeic species

[Allolobophora chlorotica (Savigny, 1826), A. leoni Michaelsen, 1891, A. dacica (Pop, 1938), A.

mehadiensis Rosa, 1895, A. hrabei (Cernosvitov, 1935), Aporrectodea georgii (Michaelsen, 1890),

Ap. jassyensis (Michaelsen, 1891), Ap. handlirschi (Rosa, 1897), Ap. rosea (Savigny, 1826), Ap.

caliginosa (Savigny, 1826), Ap. dubiosa (Örley, 1881), Proctodrilus antipai (Michaelsen, 1891), P.

tuberculatus (ernosvitov, 1935), Octodrilus transpadanus (Rosa, 1884), Oc. gradinescui (Pop, 1938),

Octolasion lacteum (Örley, 1881), Oc. montanum Wessely, 1905)] were fed cattle dung. Cast

production values in this experiment varied by an order of magnitude. Again, species differed on the

proportion of cast deposited on soil surface vs. deeper layers. The experiment highlights similarities

and differences in feeding and casting behaviour at the species level, and thus their contribution to

decomposition and nutrient cycling. This information needs to be taken into account when evaluating

changes in soil community composition and food web structure.

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Effects of different types of litter on earthworms belonging to three

ecological categories

Trigo Dolores, Berdugo M. , Novo Marta, Gutiérrez Mónica


The invasion of species could be a cause of deep changes in structure and function of native soils.

The composition of European forests is changing from monospecific formations to mixed forests

including exotic species. This fact has an influence on the physico-chemical characteristics of the soil

and on its fauna and decomposing microorganisms, by changing the nutrients cycle and the

decomposition of litter (Yelenik et al. 2007). Litter is considered as the most important source of

allelochemicals (Reigosa et al. 1999); several properties of the litter, such as a high proportion C:N,

lignin, phenols and other secondary components, are associated to a slow decomposition rate. Given

the fact that vegetal and edaphic invertebrate diversity are related to each other, the vegetal invasions

could cause important changes in the structure of the ecosystem, having an influence on the

communities and behaviour of detritivores (Mayer et al. 2005). It is known that the quality of the

litter has an influence on trophic selection, reproduction and biomass of the earthworms (Cesarz et al.

2007). At the same time, earthworms have an influence on the decomposition of the organic matter,

by improving the availability of resources for the microflora and the transfer of organic particles to

the deep layers of the soil. Any effect on the earthworms activity will indirectly influence the soil

properties. The objective of this work is to assess the effects of the litter from Acacia nigra, exotic

species in Spain, and the native species Quercus pyrenaica on the growth and reproduction of three

earthworm species belonging to the three known ecological categories Eisenia fetida, Aporrectodea

trapezoides and Hormogaster elisae. Three types of microcosms were set up (controls without litter,

treatments with acacia´s litter and problems with oak´s litter) where earthworms with a similar initial

weight were introduced. The microcosms were checked weekly. The earthworms and number of

coccons were counted and weighted. Furthermore several abiotic factors were analyzed in every

microcosm at the beginning and in the end of the experiment. Starting from the ninth week, E. fetida

exhibited a more pronounced growth when cultured in litter from oak and in the controls than when

cultured in litter from acacia. A. trapezoides growed slightly more when cultured with the two types

of litter than in the controls. No significant differences were detected in the growth of H. elisae.

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Ecosystem services of earthworms in agro-ecosystems

Tondoh Jerome E.1, Guéi Arnauth M.

2,3, Siagbé Goli

2,3, Gbakpa Edgard A.

2,3,

Baidai Yannick2,3

1CIAT-TSBF, Institut d‘Economie Rurale de Sotuba , Laboratoire Sol-Eau-Plante, BP 262 Bamako. Mali

2 UFR des Sciences de la Nature

3 Centre de Recherche en Ecologie, Université d‘Abobo-Adjamé, 02 BP 801 Abidjan 02, Côte d‘Ivoire

It is now well admitted that by their engineering activities, earthworms are able to alter key soil

properties responsible for improving soil quality and sustainability in low-input agricultural systems.

However, very few studies have investigated their impact on ecosystems services such as food

production, water infiltration capacity, soil structure regulation and carbon sequestration in agro-

ecosystems. Short-term demonstration and experimental studies based on earthworm inoculations

was undertaken in Centre-West Côte d‘Ivoire to: (i) improve maize production in farmer fields, (ii)

validate the concept of integrated soil fertility management (ISFM) using earthworms as biological

component in field mesocosms and, (ii) assess the impact of these organisms on water infiltration

capacity, soil structure regulation and carbon sequestration in field mesocosms. Results showed that

in farmer fields, earthworms are able to increase maize grain yield, cobs and biomass production

respectively at 21%, 26.9, and 27.6% relative to control and fertilizer treatments. In field mesocosms,

by significantly increasing maize phosphorus and water use efficiency 50 days after planting,

earthworms improved maize growth in treatments characterized by coffee wastes and fertilizer

applications. Maize grain rates of increase ranged from 96.3 to 266.8% in earthworm-based

treatments and from 58.8 to 111.04% in fertilizer-based treatments showing the positive impact of

earthworms in the ISFM trials. Moreover, the significant improvement of water infiltration capacity

and soil structure regulation is due to the presence of earthworms with contrasting functional

attributes in the agro-ecosystems, even if there was no significant impact of earthworms on carbon

sequestration. More field and laboratory researches are needed to confirm these preliminary

observations and promote the positive impact of earthworm in agro-ecosystems.

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Contrasting effects of soil fauna on the nitrogen dynamics of tillage and

no-tillage soils

Toyota Ayu

1,2, Frouz Jan

1, 2, Hynst Jaroslav

1, 2

1Institute of Soil biology

2Biology Centre Academy of the ASCR

Managing the soil fertility of an agricultural ecosystem for sustainable production can be

problematic. Although soil biodiversity is presumed to maintain soil fertility, the exact mechanisms

of this control have not been elucidated. Certain agriculture practices, such as no-tillage (NT) or

leguminous plant production, increase soil fauna diversity and may also improve soil nutrient

retention and plant production. NT cropping systems usually increase soil aggregation and organic

matter content relative to conventional tillage (CT). However, little is known about the separate

effects of tillage or soil biodiversity on plant production, as tillage affects both plant production and

soil biota. Hence, determining if the effect of a practice on plant production is direct or mediated by

soil biota change is difficult. In this study, we investigated the contribution of soil fauna and

agricultural practices to nitrogen dynamics, using manipulation experiments. To distinguish the

effects of NT, legume planting, and soil fauna presence, we conducted a laboratory full factorial

experiment combining CT, NT, legume (Lotus corniculatus) presence and absence, and soil fauna

presence and absence. We measured the nitrogen (N) concentration in water leaching from soil to

determine the N loss of agricultural soil systems. After 90 days, the experiment was terminated, and

the dry weight and N content of aboveground plant biomass were determined. General linear model

(GLM) analysis was used to compare the effects of soil practices, soil animal manipulations, and

their interactions on N concentration in leached water and plant biomass. Tillage significantly

increased leached nitrate and total N leached into water. While the presence or absence of fauna

alone showed no significant effect, the interaction between tillage and fauna had a significant effect

on N leaching. Faunal presence increased total N leaching in CT soil, but decreased it in NT soil. In

the faunal presence, NT soil could conserve more N than did tilled soil. The aboveground plant N

biomass of NT soil was significantly higher than that of tilled soil. Plant N was increased by fauna in

CT soil but not in NT soil. These results suggest that soil fauna in tilled soil may possibly accelerate

N cycling between the soil and plants, whereas the opposite is true of NT soil.

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Key messages for earthworm management to control soil erosion

in arable land

Valckx Jan , Govers Gerard , Hermy Martin, Muys Bart

Department of Earth and Environmental Sciences, Division Forest, Nature and Landscape, Katholieke

Universiteit Leuven, Celestijnenlaan 200E, BE-3001 Leuven, Belgium

Currently, soil erosion by water is one of the major threats on agricultural land throughout the world, and both its local (e.g. decreased crop yield) and downstream consequences (e.g. river eutrophication and

sedimentation, increased flooding risk) may intensify due to climate change. A large set of technical (e.g. non-inversion tillage); vegetational (e.g. grass buffers), structural (e.g. sedimentation pond) or land use related (e.g. set aside land) erosion control measures have been proposed, most of them expensive end-of-

pipe interventions. Up to now the potential of within-field ecological erosion control, i.e. through the action of ecosystem engineers such as earthworms, has been overlooked. The ECOWORM project (2005-2009) (Erosion control in arable land through the management of earthworm communities) tried to gain

insights in the relationships between (1) arable field management (soil cultivation, green manure), (2) earthworm biomass and species composition, and (3) sediment loss through water erosion through diverse

experiments and observations in the field and in the lab for West-European conditions. Based on these results, a toolkit was developed to inform policy makers, extension workers, and farmers about the significant role earthworms play in sustainable field management. The toolkit provides information about

the different ecological groups of earthworms and their respective functions in the soil, and gives practical advice concerning earthworm sampling, identification and interpretation of results. Also the importance of earthworms in ecological erosion control is demonstrated. Finally, the toolkit gives recommendations to

arrive at sustainable and well-considered field management, one of the key elements to successfully control erosion at the source. Seven key messages form the backbone of the toolkit: Key message 1

Within the boundary conditions set by climate and soil type, farmers‘ management is the key factor controlling earthworm abundance and species composition in arable land. Key message 2 Suitable habitat conditions (minimal soil disturbance, food of adequate quantity and quality) are the key determinant for

the restoration and maintenance of diverse earthworm communities and abundant earthworm populations. Key message 3 Reduced soil disturbance – through non-inversion tillage in particular - promotes the abundance of deep-burrowing earthworm species, of high importance for erosion control. Key message 4

Earthworm populations need one to five years to recover after years of intensive soil management, depending on initial conditions. Key message 5 Use of the widely grown cover crop Yellow mustard (Sinapis alba L.) does not harm earthworm populations. Key message 6 Earthworms - in particular deep-

burrowing species such as the night crawler (Lumbricus terrestris L.) - significantly reduce runoff and soil loss in arable land. Key message 7 Weather conditions and soil status cause large variability in the

erosion-controlling effects of reduced tillage and deep-burrowing earthworm species. These key messages underline the importance of good soil ecosystem management by the farmer and the need for the promotion of agricultural practices supporting populations of deep-burrowing earthworm species (non-

inversion tillage, direct drilling). Farmers who stimulate the occurrence of deep-burrowing earthworm species also significantly reduce the erosion risk.

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Do earthworms stimulate nitrous oxide emissions at the ecosystem level?

Whalen Joann K.1, Kernecker Maria L.

1, Prieto Roberto

1, Durocher Simon

1,

Speratti Alicia1, Sampedro Luis

2

1McGill University

2Centro de Investigacións Forestal de Lourizán

Earthworms are well known for their contribution to nitrogen mineralization, which is beneficial for

soil fertility and crop production. However, earthworms may also stimulate gaseous nitrogen losses

in terrestrial ecosystems. The earthworm gut is an anaerobic fermentation system that favours

facultative anaerobic microorganisms such as denitrifiers that produce nitrous oxide, a potent

greenhouse gas. Earthworm structures such as surface and subsurface casts (fecal pellets), middens

(soil-litter mixture at the soil surface) and burrows are also a favourable habitat for microbial

nitrifiers and denitrifiers that produce nitrous oxide. The objective of this presentation is to

summarize what we know about earthworm-microbial interactions that lead to nitrous oxide

production, to identify ecosystems that may be ―hot spots‖ for such interactions, and to determine

when are the ―hot periods‖ of nitrous oxide emission in these ecosystems. Laboratory studies

demonstrate that earthworms stimulate nitrous oxide production in plant-free soil, leading us to

hypothesize that earthworm-microbial interactions lead to nitrous oxide production at small scales

(microcosms, small field plots) and may contribute to nitrous oxide emissions at the ecosystem level.

Agroecosystems with perennial vegetation, such as grass- and legume-based hayfields, support large

earthworm populations because soils are undisturbed by cultivation and senescing plant residues are

a food source for earthworms. Moist forests, riparian buffers and floodplains are also important

because many earthworm species have adaptations that permit them to thrive in wet soils and tolerate

flooding. These ecosystems constitute ―hot spots‖ for nitrous oxide emissions attributable to

earthworm activities. The ―hot period‖ for emissions in these ecosystems may correspond to times of

maximal earthworm activity (e.g., spring and fall in temperate climates), although earthworm

structures may be a persistent source of nitrous oxide during the growing season, when climatic

conditions are favourable for microbial nitrifiers and denitrifiers. We conclude by proposing a

modelling framework to interpret small-scale biotic interactions and extrapolate them to the

ecosystem level.

156


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Adsorption and degradation of vermicompost on five phenolic acids in

watermelon soils: experiment in laboratory

Xu Yongli1, Sun Zhenjun

1, Zhang Junying

2,

1China Agriculture University

2Hebei Polytechnic University

Vermicompost can decompose some poisonous compounds, but the detailed mechanism of that on

phenolic acids is not clearly understood. Under this object, the effect of vermicompost on phenolic

acids in continuous cropping soil was detected in laboratory. Contents of five phenolic acids, 4-

hydrobenzoic acid, vanllic acid, syringic acid, ?-coumaric acid, frulic acid, extracted by water and

methanol were determined in continuous cropping soil with and without watermelon seedlings. The

proportion of sterilized and fresh vermicompost were 0%(control), 10%, 25%, 50%(volume ratio) in

mixture of continuous cropping watermelon soil and vermicompost with and without watermelon

seedlings, respectively. The data showed the amount and the concentration of phenolic acids in water

extracts were less than that in methanol extracts. So the organic extracted solution was better choice

than water for phenolic acids in soil. The methanol extracted 1-3 phenolic acids in soil while only

one by water. Sterilization of vermicompost and plant also affected phenolic acids amounts and

concentration in soil. Amount and total concentration containing sterilized vermicompost without

plant were more than those of containing fresh vermicompost, which indicated the adsorption of

vermicompost was the main mechanism in decomposition in sterilized condition. And microbe

degradation of fresh vermicompost stimulated phenolic acids decomposition, so the adsorption and

degradation was the main mechanism of decomposition with fresh vermicompost. The plant root

exudation had important effect on decomposition. The amount of phenolic acids in soil with

sterilized vermicompost with plant was less than that of without. Total concentration of phenolic

acids in soil with sterilized vermicompost with plant was more than that of without. So plant root

exudates maybe the reason that increased amounts of phenolic acids in sterilized condition. While the

concentration containing fresh vermicompost with plant was decreased. And the more decomposition

rate the more addition of fresh vermicompost. Decomposition test of pure phenolic acids in

vermicompost demonstrated the interaction of adsorption and degradation in fresh vermicompost. It

was concluded fresh vermicompost decomposed the allelopathic matters such as phenolic acids in

continuous cropping soil by adsorption of particle and degradation of microbe. The decomposition

rate was influenced by addition content of vermicompost into continuous cropping soil.

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Influence of vermicompost on seedling growth and microbial community

in watermelon monocropping soil: experiment in greenhouse

Xu Yongli1, Sun Zhenjun

1, Zhang Junying

2


2Hebei Polytechnic University

Continuous cropping system led to plant yield decrease, and pathogen increase in soil. In order to

find a biological solution to deal with continuous cropping problems, cow manure vermicompost was

chosen in this experiment for its nutrients and resistance to microbe. The tested soil was 3-year

continuous cropping watermelons. Four treatments: 0(control), 10%, 25%, 50% (volume rate), were

set. Three watermelon seedlings were left after seeds germination in each pot with 4kg of substrate

mixture in a greenhouse condition . After 40 days, plant height, stem width, leaf area and amount,

root length, biomass of shoot and root were measured. Populations of bacteria, fungi, actinomycetes,

Fusarium oxysporum were detected by plate culture. The data showed that vermicompost had more

stimulation on plant growth. For example, the plant height, stem width, leaf area, root length of

seedlings grown in mixture with vermicompost were 129.5%-147.9%, 37.0%-44.4%, 59.1%-83.9%,

61.9%-114.8% respectively higher than that of seedlings grown without vermicompost. Differences

of each factors between vermicompost treatments and control were significant Besides, reasonable

addition of vermicompost, such as 25%, was benefit for microbial community stabilization in

rhizosphere and decreased the incidence of watermelon wilt.

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Session 5

Earthworms interaction

159


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Plenary lecture – Session 5

Diversity of bacterial symbionts in earthworm nephridia

Davidson Seana K.1, Powell Ryan

1, Go Ruth

1, James Samuel

2

1University of Washington

2Kansas University

Nearly all multicellular organisms form partnerships with bacteria that confer adaptive advantages in

many different forms and environments. Earthworms are no exception and many harbour specific

bacteria within their excretory nephridia. Using a combination of microscopy and 16S rRNA gene

sequence analysis, we identified species harbouring nephridial bacteria and what types. Out of the 15

earthworm families we have screened, seven harbour nephridial symbionts. Members of the

Lumbricidae harbour Verminephrobacter spp, related to the genus Acidovorax. Although nearly all

Lumbricidae members (19 genera screened) contained nephridial Verminephrobacter, this symbiont

was not detected among the other families. However, 19 species (representing 6 families) were

positive for other types of nephridial bacteria. In addition to the Verminephrobacter spp., two or

more other bacterial types that were consistently found included a Flexibacter-related bacteria (60 %

of the Lumbricidae and in five genera outside of Lumbricidae) and Alphaproteobacteria from

Rhodospirillales (Azospirillum) and Rhizobiales. Colonization patterns suggested that both specific

symbioses and transient colonizations of certain types of bacteria occur in earthworm nephridia. The

function of these symbionts is unknown. In the lab, Eisenia fetida serves as a symbiosis model for

these widespread earthworm-bacteria associations. The entire lifecycle is easily maintained and the

Verminephrobacter is cultivable outside of the host. E. fetida transfers the nephridial bacteria, V.

eiseniae, Flexibacter and an Actinobacteria, into the egg capsules where they are recruited into the

developing embryo. Theorized functions of these symbionts include nitrogen conservation, ammonia

detoxification and chemical protection of the egg capsules. Antibiotic treatments to the eliminate egg

capsule bacteria led to fungal overgrowth suggesting that the egg capsule bacterial community may

provide protection for the capsule while it incubates in the soil. These nephridia-associated bacteria

likely influence the success of earthworms within the complex soil system.

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Oral – Session 5

Determinants of gut wall bacterial communities in earthworms

Thakuria Dwipendra1, Schmidt Olaf

2, Doohan Fiona M.

2

1University College Dublin & Central Agricultural University, India

2University College Dublin, Ireland

We investigated interactions between lumbricid earthworms and their intestinal microorganisms.

First, we tested the effects of DNA extraction and field-based earthworm preservation methods on

the quality of DNA obtained from earthworm gut contents and gut walls. We assessed several DNA

extraction methods that are known to yield high quality DNA from soil samples. Field-based

preservation methods assessed were absolute ethanol, chloroform and liquid N, with fresh

earthworms as a control. Bacterial communities were detected using automated ribosomal intergenic

spacer analysis (ARISA) of 16S and 23S genes, and ribotype data was used to assess diversity and

community composition. Second, in a series of studies, we tested the hypothesis that differences in

burrowing and feeding habits of anecic and endogeic earthworms are reflected by the existence of

specific bacterial communities attached to the gut wall. Using soil and earthworm samples collected

from adjacent wheat–barley and grass–clover fields, we found that the anecic Lumbricus terrestris

and L. friendi, the endogeic Aporrectodea caliginosa and A. longa (traditionally defined as anecic,

but shown here to possess endogeic characteristics) contain ecological group-specific gut wall-

associated bacterial communities. The abundance of specific gut wall-associated bacteria (identified

by sequence analysis of ribotype bands), including Proteobacteria, Firmicutes and an

Actinobacterium, was ecological group dependent. A microcosm experiment, conducted using A.

caliginosa and L. terrestris and five different feeding regimes, indicated that food resource can cause

shifts in gut wall-associated bacterial community, but the magnitude of these shifts did not obscure

the delineation between ecological group specificity. In the final study that used A. caliginosa and A.

longa samples collected from six different arable fields, we deduced that, within an ecological group,

habitat was a more important determinant of gut wall-associated bacterial community composition

than was host species. We conclude that the determinants of the selection of bacteria associated with

the gut wall of earthworms are in the order ecological group > habitat > species. Thakuria D. et al.,

2009. Field preservation and DNA extraction methods for intestinal microbial diversity analysis in

earthworms. J. Microbiol. Methods 76(3), 226–233. Thakuria D. et al., 2010. Gut wall bacteria of

earthworms: a natural selection process. ISME J. 4(3), 357–366.

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Oral – Session 5

Lactobacillus spp. maybe helps earthworms against dangerous pathogens

in the environment?

Liu Xuelian, Yu Yu, Yao Lan, Sun Zhenjun


When some dangerous pathogens, like E. coli O157:H7, Salmonella typhimurium and

Staphylococcus aureus were added in the living environment, six strains of Lactobacillus isolated

from the earthworm casts, which were marked as WP, HF1, HF2, 5HF, MHF, and RP respectively

and proved that these six bacteria were Gram-positive and Contact –negative and preliminarily

determined these were Lactobacillus. They were identified by 16S rDNA sequence analysis: WP as

Weissella; HF1, HF2 as Lactobacillus brevis; 5HF, MHF as Lactococus garvieae; RP as Pedicoccus

acidilacticii. These strains of Lactobacillus have broader spectrum, they show significant

antimicrobial effect on Escherichia coli, Staphylococcus aureus and Salmonella typhimurium under

certain conditions. Not only playing a role against Gram-positive bacteria, but also for Gram-

negative bacteria, so it can be considered as broad-spectrum antibacterial substance. Changes in

environmental conditions affect the antibacterial activities of the supernatants of Lactobacillus spp.,

present diversified effects to different pathogens. Changes in environmental conditions not only

affect the production of Lactobacillus and bacteriocin production, it also affects bacteriocin activities

and the adhesion to bacteria, thereby affecting the concentration and the activities of bacteriocins in

the supernatant. In stress conditions due to some dangerous pathogens, such as E.coli O157:H7,

Salmonella typhimurium and Staphylococcus aureus, Lactobacillus spp living in earthworm

alimentary canal may strengthen its antibacterial functions to help its host against those dangerous

pathogens. Much further work is needed to be done to confirm this interaction.

162


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Oral – Session 5

Biotic interaction between earthworms and pesticide degrading bacteria -

Impact on the detoxification service of soil

Monard Cécile1, Vandenkoornhuyse Philippe

1, Martin-Laurent Fabrice

2, Binet Françoise

1

1University of Rennes 1 - UMR 6553 ECOBIO

2INRA-CMSE, UMR 1229

Earthworms are efficient soil engineers that change the physical and chemical properties of soil and

act in turn on soil microbial communities. Earthworm bioturbation will thus impact microbial

processes in soil and also the generated ecosystemic services such as detoxification function. By

using RNA based Stable Isotope Probing we demonstrated that the bacterial communities responsive

to 13

C enrichments in earthworm casts were different from those in bulk soil resulting in different

degradation kinetics of 13

C labelled substrates. From these results we focused on the impact of

earthworm soil bioturbation on pesticide degradation by using atrazine as a model. This pesticide has

been widely used leading to soil bacteria adaptation to its degradation and the degradative pathway

involved has been elucidated (atzA, B, C, D, E and F genes). We hypothesized that earthworms, by

generated spatial heterogeneity, modify the abundance and activity of atrazine-degraders in the soil

microsites they engineer and act on the atrazine depollution service sustained by the adapted soil

bacteria. Atrazine degradation genes as well as their expression were quantified by qPCR during in

situ degradation of atrazine in the soil drilosphere (burrow-linings and casts) and bulked samples

from two soils with different abilities to mineralize atrazine. The kinetic of 14

C-atrazine

mineralization was measured to link the atrazine-degraders abundance and activity changes to the

degradation function. We observed different results depending on the initial soil but the atrazine

mineralisation was always related to the atrazine degradation gene amount and expression. In the soil

that slowly mineralised atrazine, we observed a positive ‗‗burrow-lining‘‘ effect on atrazine degrader

abundance and activity compared to bulk soil whereas a negative impact was detected in earthworm

casts. In the soil that quickly mineralised atrazine, opposite results were obtained. It seemed that

depending on the soil physico-chemical properties and the inhabiting bacterial communities the

impact of earthworm soil bioturbation on atrazine mineralization was different. However, in both

soils atrazine mineralization was increased in certain microsite generated by earthworm bioturbation

indicating their special feature regarding microbial activity and thus pollutant degradation.

163


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Oral – Session 5

Interaction between earthworms and soil fungi: volatiles attraction

Zirbes Lara1, Thonart Philippe

2, Wathelet Jean-Paul

3, Haubruge Eric

1

1Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liege, Gembloux,

Belgium 2Laboratory of Bioindustries, Gembloux Agro-Bio Tech, University of Liege, Gembloux, Belgium

3Laboratory of General and Organic Chemistry, Gembloux Agro-Bio Tech, University of Liege, Gembloux,

Belgium

Earthworms and microorganisms are two major representatives in soil. Several interactions are

known between these both actors of soil. But principally, microorganisms are an unavoidable

constituent of earthworms‘ diet, in particular soil fungi are assumed to be a major food source for

earthworms. Some fungi are presents in guts and casts of earthworms whereas some others are

completely digested. For example, Eisenia fetida totally digests the fungi Geotrichum candidum and

not Aspergillus fumigates. Geotrichum candidum is found in various habitats such as the soil and is a

saprophyte in gut of humans and other animals as earthworms. Eisenia fetida is an important

ecological earthworm species that is commonly used in industrial vermiculture and vermicomposting

and is the model species for all scientist ecotoxicological researches. Chemical ecology and close

association with soil fungi of this earthworm are poorly study. Therefore this information is essential

to understand E. fetida life and to enhance our biological knowledge. As a first step to achieve such

understanding, we focus our study on the behaviour of E. fetida in presence of filtrate of G. candidum

culture in 4-arm olfactometer. We show for the first time, that E. fetida is attracted by filtrate of G.

candidum culture. Volatile molecules emitted by this filtrate are analysed and identified by SPME-

GCMS. Each identified molecule is tested in 4-arm olfactometer in order to find which are

responsible for E. fetida attraction. We find that this attraction is due to ethyl pentanoate and ethyl

hexanoate.

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Oral – Session 5

Earthworm - denitrifier interactions play a role in increased nitrous oxide

emissions from soil mesocosms amended with crop residue

Nebert D. Lucas, Bloem Japp, Lubbers Ingrid, Van Groenigen J.W.


Recent studies suggest that earthworm activity increases emissions of nitrous oxide (N2O), a potent

greenhouse gas, from arable soils. Understanding this mechanism is of great importance to

sustainable agricultural management. Earthworm feeding strategy has been suggested as a driving

factor, as it determines their access to fresh organic carbon for denitrification. Here, we determined

whether interactions between earthworm feeding strategy and the soil denitrifier community can

predict N2O emissions from the soil. We set up a 90-day mesocosm experiment in which 15

N labeled

maize (Zea mays L.) residue was applied either on top or manually incorporated into the soil;

additional treatments included presence or absence of the epigeic earthworm species Lumbricus

rubelllus and/or the endogeic earthworm Aporrectodea caliginosa. On days 6, 21, and 90 we tested

the bulk soil for denitrification enzyme activity as well as the abundance of total bacteria (16S rRNA)

and denitrifier genes nirS and nosZ through real-time qPCR. Cumulative N2O emissions after 90 days

were significantly enhanced by the presence of L. rubellus (76%; p = 0.014) in the top-applied

treatment. There was no significant effect of A. caliginosa, nor a species interaction on cumulative

N2O after 90 days. However total emissions in the treatment with both species had comparable

emissions to mesocosms with manually incorporated residue (159.9 vs 156.3 mg N2O-N kg-1

soil,

respectively). On day 21 A caliginosa presence significantly increased N2O emissions (117%; p =

0.005) and decreased microbial biomass (-28%; p = 0.004). The presence of L. rubellus caused a

significant increase in denitrification enzyme activity in top-applied residue treatments on days 21

and 90 (p = 0.034 and 0.001, respectively). No changes in 16S rRNA or nirS abundance were

observed. However, total nosZ abundance was significantly increased (100%; p < 0.05) on day 90 in

the top-applied residue treatment containing only L. rubellus. On day 21 the presence of L. rubellus

significantly decreased the proportion of soil bacteria containing the nirS gene (p = 0.010), indicating

a possible shift in the denitrifier community.

We conclude that the effect of L. rubellus on increased N2O emissions is related to its ability to

enhance denitrifier community activity and affect composition through incorporation of fresh organic

matter into the soil. The A. caliginosa effect appeared to be linked to an increase in general microbial

turnover.

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Oral – Session 5

Using molecular diet analysis to evaluate the role of earthworms

in food webs

Boyer Stephane1, Wratten Stephen D.

1, Holyoake Andrew

1, Cruickshank Robert H.

2,

Abdelkrim Jawad3

1Bio-Protection Research Centre, Lincoln University New Zealand

2Agriculture and Life Sciences Division, Lincoln University, New Zealand

3Centre for Reproduction and Genomics, Department of Anatomy & Structural Biology, University of Otago,

New Zealand

Ecosystem services provided by earthworms include increasing soil fertility and recycling organic

material. As well as their key role in improving soil structure and chemistry, and their overall

positive influence on plant communities, earthworms often represent a substantial biomass, which

supports large communities of vertebrate and invertebrate predators. Estimating the role of

earthworms in food webs is essential for a thorough measurement of the ecosystem services they

provide. This requires identifying all predators feeding on earthworms. Because their soft body is

rapidly digested, the assessment of earthworms in predators‘ diet often relies on the detection of

chaetae in the faeces of the predators. But earthworm chaetae are not species-diagnostic and which

species is predated remains largely unknown. The development of molecular techniques targeting

prey DNA remaining in predators‘ guts or faeces is a promising tool for identifying earthworm

species. The techniques used here are usually based on the polymerase chain reaction (PCR). This

study proposes a method for species identification of predated earthworms using predator faeces. We

conducted molecular analyses on the faeces of a carnivorous landsnail endemic to New Zealand

(Powelliphanta augusta). Earthworm group-specific primers and next-generation sequencing (454-

pyrosequencing) were used to extract and sequence earthworm DNA. Although earthworm tissue

was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity for

molecular tools to detect and analyse it. Results showed that the studied landsnails feed on at least six

species of New Zealand endemic earthworms. The method developed here is particularly appropriate

for investigating the diet of potential predators for which feeding behaviour is difficult to observe.

Such studies are required to draw a complete picture of the ecosystem services provided by

earthworm communities. In this case it also helps conservation of rare and endangered predator

populations.

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Oral – Session 5

Importance of earthworm – seed interactions for the composition of plant

communities: a review

Decaëns Thibaud1, Forey Estelle

1, Clause Julia

1, Scheu Stefan

2, Barot Sebastien

3,

Langlois Estelle1, Margerie Pierre

1, Kam-Rigne Laossi

4, Eisenhauer Nico

2

1Laboratoire ECODIV - Univ of Rouen (France)

2J.F. Blumenbach Institute of Zoology and Anthropology - Univ of Göttingen (Germany)

3UMR 7618 Bioemco - IRD Bondy (France)

4UMR 7618 Bioemco - IRD Bondy (France).

Soil seed banks are crucial elements for the understanding of plant population and community

ecology. Among animal species, earthworms are increasingly recognised as important dispersers and

predators of seeds. Through direct and indirect effects, they might affect either positively or

negatively the distribution, survival and establishment of seeds in plant communities. Thereby,

earthworms may affect seedling establishment by a variety of mechanisms such as selective ingestion

and digestion of seeds, acceleration or delaying of seed germination, and downward or upward seed

transport. Seed displacements might for instance reduce exposure to harsh environmental constrains

and to aboveground seed predators. Surface earthworm casts and middens often contain many viable

seeds and might be important regeneration niches for plant seedlings and therefore likely favour

specific plant strategies. Nevertheless, the role of earthworm as seed dispersers and mediators of seed

bank dynamics has seldom been considered in past studies, especially in case of natural plant

communities. Many aspects of the processes induced by earthworm activity remain obscure. Thus,

we propose in this review to discuss the potential consequences of earthworm-mediated impacts on

soil seed banks, plant community dynamics and potential outlooks in plant evolution, restoration, and

conservation ecology.

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Oral – Session 5

Secondary seed dispersal of Ambrosia trifida by Lumbricus terrestris:

Implications for Earthworm – Weed Interactions

Harrison Kent1, Regnier Emilie

1, Jianyang Liu

2, Arancon Norman

3, Edwards Clive A.

1

1Ohio State University

2University of Illinois

3University of Hawaii – Hilo

Lumbricus terrestris L. is a naturalized earthworm in North America that actively collects and caches

seeds of Ambrosia trifida in its burrow. As a native annual plant of North America, A. trifida has

become an important agricultural and allergenic weed of the U.S. within the past 30 years. Burial of

A. trifida seeds in L. terrestris burrows reduces seed exposure to predators and influences depth-

dependent seed germination and seedling recruitment. We conducted field experiments to determine

the interaction of L. terrestris and vertebrate seed predators on A. trifida seed fate and seedling

recruitment in different habitats. A Bayesian model was fit to the data to assess the effects of L.

terrestris on A. trifida in the presence and absence of seed predators. Burial of A. trifida seeds by L.

terrestris increased the probability of seedling recruitment in areas where seed predation intensity

was high, but decreased the probability of recruitment when predators were excluded or when

predation intensity was low. In areas with high vegetative cover, seed predators consumed more

seeds than were cached by L. terrestris. Conversely, L. terrestris cached more seeds than were

consumed by seed predators in areas with low vegetative cover. Results from field studies provide

evidence that the relationship between L. terrestris and A. trifida is a conditional mutualism wherein

L. terrestris benefits A. trifida survival by increasing seedling recruitment in habitats where seed

predation intensity is high. Furthermore, seed caching by L. terrestris may also increase A. trifida

survivorship by burying some seeds below the germination depth limit, thereby maintaining seed

dormancy and increasing seed bank longevity. Laboratory experiments designed to investigate L.

terrestris nocturnal foraging behavior revealed that the earthworms preferentially collected real seeds

over plaster-cast seed duplicates and debris, and that they were able to detect and forage the seeds

from below as well as above the soil surface. This feeding behavior suggests that the earthworm

utilizes olfactory sensing to forage seeds surreptitiously, thus limiting its exposure to predators while

promoting the mutualism with A. trifida.

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Oral – Session 5

Do non-native earthworms cause invasional meltdowns?

Cameron Erin K., Bayne Erin M.

University of Alberta

Invasional meltdowns, in which facilitation between species causes an accelerating increase in the

number of introduced species or their impacts, can result in larger changes to invaded systems than

otherwise expected. Earthworm invasions of northern forests have been suggested as an example of

an invasional meltdown, with epigeic and epi-endogeic species altering soil structure and facilitating

the establishment of endogeic or anecic earthworms that may be less capable of invading intact forest

floors. We used intact boreal forest soil cores to examine facilitation between an epigeic species

(Dendrobaena octaedra) and an anecic species (Lumbricus terrestris). Cores were inoculated with

either L. terrestris alone or both L. terrestris and D. octaedra and maintained in a greenhouse. After

3.5 months, we determined earthworm abundance, biomass, cocoon production, and organic horizon

depths in the different treatments. There were no significant differences in L. terrestris survival,

biomass, or average cocoon production between treatments. As well, the two species appeared to

have an additive rather than a synergistic effect on organic horizon depths. These results suggest that

D. octaedra does not facilitate reproduction or survival of L. terrestris and that the two species do not

cause a synergistic effect on leaf litter as would be expected in an invasional meltdown. Since D.

octaedra does not appear to greatly increase the success of L. terrestris, management in boreal forest

ecosystems may be most effective if focused on L. terrestris, which has larger impacts on its own

than D. octaedra.

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Oral – Session 5

Spatial distribution of earthworm species assemblages in pasture of

northwestern France

Richard Benoit1, Legras Marc

2, Mathieu Jérôme

3, Decaëns Thibaud

1.

1Laboratoire ECODIV - Univ of Rouen (France)

2Laboratoire BIOSOL - ESITPA Rouen (France)

3UMR BIOEMCO - Univ Paris VI

Earthworms represent one of the more important groups of soil fauna in many terrestrial ecosystems.

They are involved in the regulation of critical soil processes and in the providing of key ecosystem

services. Understanding the spatial patterns of species assemblages and their impact on soil

functioning at different scales has become an important goal in soil ecology. Earthworm populations

often exhibit conspicuous patterns, specimens being aggregated in patches which relative

arrangement responds to both habitat constraints and species interactions. In temperate regions, these

patterns have been the subject of a relatively low number of studies, particularly in context of

agricultural pastures. In this study, we investigated the spatial distribution of lumbricid earthworm

assemblages at the scale of pastures plots of different ages (one permanent pasture and two

temporary pastures with different agricultural management). Study site was located in Upper-

Normandy in France. Earthworms were collected each 10m on a regular grid and identified at species

level. Data were analysed with multivariate and geostatistical tools in order to assess if earthworm

communities will present some typical patches distribution. At this moment data are still under

analysis. Our main hypothesis is that species assembly rules during pasture progressively drives

assemblages towards a dynamic-equilibrium stage in which competitive interactions are reduced by

niche complementary and spatial segregation.

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Seed consumption and burial by the earthworm Lumbricus terrestris

favours the establishment of Lolium perenne over Agrostis capillaris

Aira Manuel1, Trevor Piearce

2

1Universidad de Vigo,

2Lancaster University

We studied the effects of the anecic earthworm Lumbricus terrestris L. on seed germination of two

plant species to understand the mechanisms underlying earthworm–seed interactions. We set up

microcosms with seeds of Lolium perenne L. (large seeds) and Agrostis capillaris L. (small seeds), in

a loam based compost, with and without earthworms. We buried seeds at three depths and recorded

their germination, we also examined to what depth earthworms buried seeds, and we studied seed

viability after transit through the gut of earthworms. Earthworms dramatically reduced the numbers

of A. capillaris seeds germinating, but did not modify the germination of L. perenne seeds.

Earthworms, however, increased the biomass of L. perenne seedlings. When we buried the seeds,

those from A. capillaris rarely germinated when they were placed below 7 cm, unlike L. perenne

seeds. Earthworms buried the seeds of L. perenne to a depth of 15 cm. Passage of seeds through the

gut of earthworms had a detrimental effect on A. capillaris seed viability, while L. perenne seeds

remained unaffected. While our results show that earthworm activity can strongly influence grass

seed germination and plant establishment in microcosm experiments, further work is needed to assess

the implications of earthworm–seed interactions for vegetation dynamics in the field.

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Pre-copulatory and male reproductive allocation in relation to inbreeding,

outbreeding and non size-assortative mating in the earthworm Eisenia

andrei (Oligochaeta: Lumbricidae)

Aira Manuel, González Porto Pablo, Domínguez Jorge

Universidad de Vigo

In simultaneous hermaphroditic animals like earthworms, the division of reproductive resources into

male and female function is an important strategy for optimizing fitness. Thus, the earthworm

Eisenia andrei (Oligochaeta: Lumbricidae) reduces cocoon production under inbreeding, outbreeding

and non size-assortative mating. However, we do not know whether this reproductive adjustment

(post-copula) comes from a female effort decision or from a decrease in the sperm received (i.e. male

effort decision). In order to clarify this question we tested whether the genetic similarity (inbreeding

and outbreeding) and the relative size of partners influence the volume of sperm donated. We also

explored the possibility of pre-copula adjustment via mate choice decision. Mate choice decision was

evaluated setting up mating trios in which a focal individual can choose between other two

earthworms: one sibling, and one unrelated (inbreeding); one from the same population, and one

from a different population (outbreeding); and one of the same size, and one of a different size (size-

assortative). Male effort decision, measured as the volume of sperm donated, was studied setting up

the following mating pairs: inbreeding pairs, mating two fullsiblings and two unrelated earthworm

from the same population; outbreeding pairs, mating an earthworm with another earthworm from a

distant or the same population; and size-assortative pairs, mating an earthworm with another of

different or similar weight. Our results revealed that: (1) inbreeding but not outbreeding affects to the

mate choice decision reducing the probability of copula, (2) neither inbreeding nor outbreeding

affects to the volume of sperm donated during mating, and (3) earthworms have a trend to a non size-

assortative mating. Thus, small earthworms prefer to mate with large partners whereas large

earthworms are less choosy. Further, small earthworms donate more sperm to large than to small

partners, however large earthworms do not significantly change the volume of sperm donated.

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How to reduce the number of Meloidogyne paranaensis galls in tomato

using earthworms?

Alves Dionicio Jair, Arlei Marcela, Lunardi Marilene de Fátima

Universidade Federal do Paraná

The objective of the study was to evaluate the incidence of Meloidogyne paranaensis galls in

Solanum lycopersicon roots after inoculating Amynthas sp. and Pontoscolex corethrurus. The

experiment was conducted in the greenhouse of the Nematology Laboratory of ―Marcos Enrietti‖

Diagnosis Center of the Department of Agriculture and Supply of the state of Paraná (SEAB), using

randomized blocks, with eight treatments and four replicates: T1- standard; T2-Meloidogyne

paranaensis; T3-Amyntas sp.; T4-Pontoscolex corethrurus; T5- Amynthas sp. + M. paranaensis; T6-

P. corethrurus + M. paranaensis; T7- Amynthas sp. + P. corethrurus; T8- Amynthas sp. + P.

corethrurus + M. paranaensis. Initially, six adult of Amynthas sp. or P. corethrurus earthworms were

inoculated, individually or in the same proportion (3:3) with fresh weight previously determined, in a

4L polyethylene pot containing three litres of sterilized soil. After one week, tomato seedlings

(cultivar 'Rutgers') were transplanted into the pots and 5 mL of egg suspension of M.paranaensis

containing 5,000 eggs and/or juveniles per pot were inoculated. At 65 days after inoculation, they

were assessed the number of remaining earthworms by hand-sorting, the fresh weight by direct

weighing, and the number of galls on tomato roots by direct counting using a stereoscopic

microscope. The results showed a decrease in the number of galls/plant in those treatments where

worms were inoculated, ranging from 44.8% to 60.8% for Amynthas sp and P. corethrurus,

respectively. Yet, the incidence of galls decreased 54% with the combination of earthworms. The

remnant rate of worms ranged from 16.7% to 62.5%, with higher values for the genus Amynthas.

However, understanding the interaction ―nematodes x earthworms‖ need to be studied further.

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Genetic identification of bacteria isolated from the digestive tract of the

earthworm Pontoscolex corethrurus

Brito Vega Hortensia1, Espinosa Victoria David

1, Barois Isabelle

2, Lavelle Patrick

3,

Gómez-Vásquez Armando4

1Colegio de Postgraduados

2Instituto de Ecología, A. C.

3Université Paris VI

4Universidad Juárez Autónoma de Tabasco

Invertebrates and soil microbes establish interactions or relations through which they make use of minerals and organic resources. There is little knowledge of the interaction which takes place between

earthworms and soil bacteria. The bacteria are transported to the inside of the digestive tract when these invertebrates ingest soil. It is known that within the intestines, bacteria degrade complex organic compounds such as cellulose, lignin, tannins and humic complexes, by means of enzymes which they

secrete. The objective of this study was to genetically characterize the populations of bacteria which inhabit the digestive tract of the earthworm Pontoscolex corethrurus. The sampling of the species P.

corethrurus was carried out in two localities within the state of Veracruz, Mexico: ―Plan de las Hayas‖, which bears cattle livestock pastures and ―La Mancha‖, a conservation zone with a section assigned to banana production. The earthworms were superficially sterilized, and under aseptic conditions, the

intestinal content was placed in a Brain Heart Infusion (BHI) liquid medium. The resulting suspension was inoculated in Petri dishes containing nutrient agar, Tripticasein soy agar, King medium, Pseudomonas and BHI agar. DNA was extracted from the purified bacterial isolations for PCR amplification of the

region corresponding to the 16S rDNA gene. The amplified region was partially sequenced with the aim of identifying bacterial isolates. A total of 8 bacterial genera were identified within the digestive tracts of

P. corethrurus isolated from both sampling zones: Bacillus, Bacterium, Terribacillus, Pseudomonas, Acinetobacter, Massilia, Aeromonas and Citrococcus. The sample taken from ―Plan de las Hayas‖ presented 11 bacteria species: Pseudomonas aeruginosa, Massilia timonae, Bacterium sp, Acinetobacter

sp., Aeromonas sp., Citrococcus sp., Bacillus sp., Bacillus subtilis subsp. subtilis, Bacillus mycoides, Pseudomonas sp., and Bacillus cereus. In contrast, 6 bacteria species were identified from ―La Mancha‖: Bacillus sp., Bacillus megaterium, Bacillus horikoshii, Aeromonas punctata, Bacterium, and Terribacillus

sp. The most predominant genus was Bacillus with 5 species in both sampling sites from the digestive tract of P. corethrurus: Bacillus megaterium, B. horikoshii, B. mycoides, B. subtilis subsp. subtilis and B. cereus. The present research demonstrates the existence of the genetic diversity of bacteria which inhabit

the digestive tract of the earthworm P. corethrurus. It is possible that this bacterial diversity is due to the soil type and management. The ―La Mancha‖ site presents sandy soils with banana cultivation, meanwhile

―Plan de las Hayas‖ has clayed soils which supports pastures. Future research should focus in knowing the transfer time and permanency of the bacterial species in the different parts of the digestive tract of the earthworms, likewise determine the role of bacteria in their passage through the intestines of these

invertebrates.

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Influence of body size and geographic distance in reproductive isolation of

the genus Balanteodrilus (Acanthodrilidae, Oligochaeta)

Campos Elizabeth

1, Fragoso Carlos

2

1Facultad de Ciencias Universidad Nacional Autónoma de Mexico

2Instituto de Ecología A.C.

Balanteodrilus pearsei is a native species widely distributed along southeast Mexico. In contrast to

the majority of Mexican native species, its distribution has not been reduced by human activities,

probably due to its high adaptative plasticity and wide morphological variability. This variability is

partially expressed by different patterns of genital markings present in several populations and, more

conspicuously, in the length of body size. Two morphs have been recognized that strongly differ in

the last characteristic: a small, narrow distributed morph mainly restricted to sandy soils, and a larger

one, widely distribute and with no clear soil texture restrictions. The aim of this study was to evaluate

reproduction between two populations of each one of the two morphs, located at different

geographical distances. Laboratory cultures of intrapopulation and interpopulation treatments were

carried out with virgin individuals from each population and morph. Reproduction was evaluated by

the number and viability of produced cocoons. Preliminary results reveal that the small morph is

reproductive isolated from the larger one, and also, that reproductive success within the same morph

is inversely related to geographic distance. As far as reproductive success is concern, this study

brings out ecological evidence that suggests that the small morph is reproductive isolated from the

larger one, and thus that it represents a valid (new) biological species. Further anatomical and

molecular studies should support this founding. Finally it is discussed the importance of size and

distance in the separation of congeneric earthworm species.

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Importance of direct earthworm-seed interactions along a succession

gradient in chalk grasslands

Clause Julia, Margerie Pierre, Langlois Estelle, Decaëns Thibaud, Forey Estelle

University of Rouen (France)

Many researches showed the beneficial role of earthworms in plant productivity through their

positive impact on soil physical, chemical and biological properties. Nevertheless, the role of

earthworms on the composition and the dynamic of plant communities had seldom been considered

in past studies. Many aspects of the processes induced by earthworm activity on natural community

remain obscure. This study focuses on the interactions between earthworms and seeds. The leading

question is: do earthworms affect the composition and the dynamic of chalk grasslands via their

direct effects on the seed bank? Three hypotheses structured the study: (i) Earthworms (EW) modify

the repartition of seeds by deeply burying seeds and by taking buried seeds out to the soil surface, (ii)

the ingestion of seeds is ―seed-EW‖ dependent and related to species traits. This ―seed-EW‖

specificity favours and/or inhibits the germination of some species, (iii) the extent of these EW-seeds

interactions varies along the secondary vegetation succession. The study site was located on chalk

grasslands of Upper-Normandy (North-West of France) on a diversity-rich protected site. This

fragmented habitat is partly managed by grazing and that is part of the regional patrimony. Soil and

earthworm casts –that contain seeds- were sampled in four different stages of the secondary

succession from open grasslands to bushes in order to create a gradient. Each soil sample was divided

into three depths (0-2 cm; 2-5 cm; 5-10cm) to evaluate each seed composition and to compare it with

cast seed composition. Species were identified at the seedling stage. Naturally expressed vegetation

was later identified in situ. Results were analysed using multivariate techniques (Co-inertia Analysis)

to detect the effect of EW on seed banks composition and dynamic. Additionally, seed selectivity by

earthworms and effects on germination success were tested in microcosms. Fifteen plant species

identified on-site and/or in EW casts and two EW species commonly found in the site were selected:

Allobophora chlorotica and Aporrectodea caleginosa. Preliminary results of this study suggest that

the similarity between soil seed banks and casts changes along the successional gradient. We will

discuss the potential consequences of EW-seeds interactions on soil seed banks, plant community

dynamics and potential outlooks in conservation ecology.

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Earthworm communities along an altitudinal gradient in Central Mexico

highest mountain: Influence of local soil type, regional weather and human

disturbance.

Cué Karina, Fragoso Carlos, Angeles Antonio

Instituto de Ecología A.C.

High mountains are important as endemic hotspots and as a refugee for native species. Central

Mexico is characterized by a high altitude plateau (2000 m over sea level), with several isolated

peaks over 3000 m of altitude. The ―Pico de Orizaba‖ region (PO), currently the highest peak in the

country (5675 m), belongs to this group of mountains within the Mexican Transvolcanic Belt.

Hitherto, Mexican earthworms have been well studied in lowlands of the southern region, but there

are few records in the central mountains, which are expected to harbour a rich still unknown

diversity. In this study we characterized earthworm communities (species richness, abundance,

biomass, ecological categories, origin and diversity) along four altitudinal gradients in PO (2000-

3500 m), two located in the east-humid slope and two in the west-more dry slope. In each transect

four sites were sampled at varying altitudes. Vegetation types included pine, oak and fir forests, high

altitude grasslands and cultivated fields. In each site qualitative (five monoliths of 0.25 m2 surface

and 20 cm depth) and qualitative methods were used. Results indicate that exotic earthworms were

mainly found in altitudes below 3000 m, in oak forests, cultivated areas and pine tree plantations,

whereas natives were limited to undisturbed forests and grasslands at or over 3000 m of altitude.

Abundance and diversity were higher in the east-humid mountainside. We discuss if the narrow

altitudinal gradient were natives were found, is a natural pattern or a response to local human

disturbance, exotic invasions or global climate change.

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Predominant Microbial Populations Associated with Earthworms Living

close to Extreme Environments of Volcanic Origin: A Comparison

Between Azorean and Mexican Populations of Pontoscolex corethrurus

Cunha Luis1, Hernández Gustavo

2, Aguiar Paula

3, Barois Isabelle

4, Morgan John

5,

Simoes Nelson1, Rodrigues Armindo

1, Montiel Rafael

2

1Centro de Investigação de Recursos Naturais (CIRN), Universidade dos Açores, Ponta Delgada, Portugal;

Langebio 2CINVESTVAV, Irapuato Gto. Mexico

3University of Azores

4Instituto de Ecología, A.C., Xalapa, Veracruz, Mexico

5Cardiff School of Biosciences, Cardiff University, Wales, UK

The extant volcanic activity in certain terrestrial locations in São Miguel, one of the nine islands

comprising the Azores archipelago, provide good ―field-laboratories‖ to study life in extreme

environments of volcanic origin. Microorganisms are believed to play an essential role in

earthworm‘s adaptation process, especially the ones passing through the guts and thereby facilitating

digestion among other features. In this study we compared by using a genomic approach the

dominant bacterial populations present in two distinct populations of P. corethrurus, one living

inside an hydrothermal field in Azores and the other, a population resident in pastures in Veracruz,

Mexico. For each studied population a genomic library was created and clones were successfully

sequenced and analysed. There were few numerically dominant species as well of intermediated

abundance but a large number of rare species. In both genomic libraries the microbial relative

abundance was dominated by the subclass of the class Proteobacteria. Azorean population was found

to have higher species richness and diversity than those from Mexico. The most dominant genera in

Mexican samples were found to be Bradyrhizobium, Methylobacterium, Nitrobacter, Paracoccus,

Pectobacterium, Rhizobium, Rhodopseudomonas, Burkholderia, Pseudomonas, Stenotrophomonas,

Blattabacterium, Dickeya, Edwardsiella, Escherichia and Serratia. In Azorean populations dominant

microbial groups belonged to genera such as Agrobacterium, Azorhizobium, Bradyrhizobium,

Caulobacter, Mesorhizobium, Methylobacterium, Methylocella, Nitrobacter, Rhizobium,

Rhodobacter, Rhodopseudomonas, Acidovorax, Bordetella, Burkholderia, Verminephrobacter,

Anaeromyxobacter, Desulfovibrio, Haliangium, Acinetobacter, Pseudomonas, Xanthomonas,

Arthrobacter, Clavibacter, Conexibacter, Corynebacterium, Eggerthella, Serratia, Cyanothece and

Gloeobacter. The implications of possible mutualistic relationships and the different metabolic

groups are further discussed.

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The role of bacterial swimming and twitching motility during recruitment

of earthworm symbionts

Davidson Seana K., Dulla Glen, Stahl David A.

University of Washington

Earthworms harbor bacterial symbionts in their excretory nephridia. Most members of the

Lumbricidae contain a Verminephrobacter species in addition to one to three additional bacterial

types. Isolates of Verminephrobacter eiseniae from Eisenia fetida have been brought into cultivation

and a full genome sequenced and annotated. Targeted genetic transformation of the V. eisenia enable

studies of genes, mechanisms and functions involved in this host-symbiont system. The nephridial

symbionts are deposited into the egg capsule albumin by the parent, and bacteria colonize the

embryos beginning mid development. Recruitment of the bacteria to the specific tissue sites is a

multi-step process beginning with aggregation at a pore, and migration of bacterial cells into a canal

leading to the nascent nephridia. We hypothesized that motility was required for early recruitment of

the symbionts. To begin to elucidate bacterial recruitment mechanisms, we targeted flagellar motility

and twitching motility for study. Twitching motility uses pili, anchored in the cell membrane, that

extend from the cell to bind a target then retract to pull the cell forward. The pilus tip often possesses

adhesins for specific binding to targets. Using the suicide vector pLVCD for directed mutagenesis,

we generated flagellar (flgK-) and type IV pilus (pilBC-) mutants of V. eiseniae. Disruption of V.

eisenia pilus genes eliminated synthesis of type IV pili, twitching motility and aggregation of cells in

liquid medium. Mutation of the flgK gene eliminated flagellar synthesis, resulting in loss of

swimming motility. We tested these mutants for their ability to colonize E. fetida embryos by

injecting bacterial cells into egg capsules lacking their V. eiseniae symbionts, and waiting for

embryos to develop. The flgK- mutants were able to migrate partially, but not completely, into the

nephridia. The pilBC- mutants did not appear in any portion of the hatchling nephridia. These

findings suggest that surface adhesion is critical for initiation of this association, and cell surface

recognition may play an important role for selection of the proper bacteria. The ability of non-

swimming V. eisenia to migrate partially, but not completely, indicates swimming motility is not

necessary for the initial entrance, but is needed to complete the journey. Future studies will

investigate the targets of the adhesins also found in the V. eiseniae genome that likely direct

symbionts to their specific colonization sites.

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Life cycle and reproductive biology of the earthworm Allolobophoridella

eiseni (Oligochaeta, Lumbricidae)

Domínguez Jorge, González Porto Pablo, Aira Manuel

Universidad de Vigo

The earthworm Allolobophoridella eiseni (Oligochaeta: Lumbricidae) is an epigeic species often

found underneath leaves or rotting trunks and is widespread in Western Europe. In order to determine

the characteristics of its life cycle, earthworms of this species belonging to three different populations

were cultured from hatchling until maturity in isolation. Development rates, cocoon production, and

incubation period were recorded under controlled conditions. On average, individuals of A. eiseni

reached maturity after day 43, with a weight ranging between 175 and 184 mg. Virgin earthworms

started to lay cocoons after reach maturity indicating uniparental reproduction. Cocoon production

was 7 cocoons per month, the incubation period was 24 days, hatching 3 individual per cocoon, with

a mean hatchability of 80%. Copulation occurs in the field; we found several individuals (35% of

captured earthworms) carrying spermatophores with spermatozoa. Our results showed that the

characteristics of the life cycle of A. eiseni are typical of an epigeic earthworm species; it means high

growth rates and high fecundity. More studies are necessary to identify what kind of uniparental

reproduction this species has and to discover how and under what conditions copulation takes place.

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Understanding the fate and linkage of N and S in earthworm engineered

peat soil using NanoSIMS

Gicquel Aurélien1, France André-Jean

1, Delhaye Thomas

2, Gruau Gérard

3,

Hallaire Vincent4, Binet Françoise

1

1UMR CNRS 6553 ECOBIO / University of Rennes 1, France

2NanoSIMS Platform of Rennes / University of Rennes 1, France

3UMR CNRS 6118 Géosciences Rennes / University of Rennes 1, France

4UMR INRA 1069 SAS / Agrocampus Ouest, France

Earthworms, by changing soil physical properties, are efficient engineers that play a key role on the

soil nutrient dynamics but their biological processes at the micrometric scale are still misunderstood.

The nano-scale secondary ion mass spectrometry (NanoSIMS) is a new tool in the study of

biophysical interfaces in soils with its ability to operate at high mass resolution, while maintaining

both excellent signal transmission and high spatial resolution (down 50 nm). Based on this new

technology, the aim of this study was to visualize at the micrometric scale the burrow-linings of

Lumbricus rubellus L. and to determine the fate and linkage of nitrogen (N) and sulphur (S) deriving

from this soil engineer. Earthworms were first triple labelled with isotopic tracers (15

N and 34

S) prior

to introduction into unlabelled peat mesocosms for 24 days. Then, the mesocosms were embedded in

a polyester resin to carry out the NanoSIMS analysis (NanoSIMS 50™; Cameca, Gennevilliers,

France). Burrow-linings were analysed with a transect of images increasingly far of the biophysical

interface (from poral space to inner of the burrow-lining) and we assessed the linkage of 15

N and 34

S

with the soil micro-architecture by using the linescan technique. Isotopic compositions were

determined by using image processing with the ImageJ software (W.S. Rasband, US National

Institutes of Health) and thus nutrients transfers from labelled earthworms to soil burrow-linings

were detected. We had a visible gradient with a decrease of percentage of 15

N and 34

S (2.00 to 0.38 %

and 10.0 to 4.4 % respectively) and no transfer was detected beyond 1mm, which defined clearly the

area of earthworm‘s influence (i.e. the drilosphere). Interestingly, the occurrence of spatially anti-

correlated hotspots of 15

N and 34

S suggested either i) two kinds of earthworm‘s excretions (an

excretion rich in N and poor in S, and an excretion poor in N and rich in S) or ii) two kinds of

microbial activity linked with N or S cycle (15

N or 34

S microbial uptake). The speciation of these

enriched hotspots are currently under analyzes. By using the NanoSIMS tool coupled with isotopic

tracers, we bring out new understandings of the impact of earthworms on the nutrients and thus their

ability in sustaining hot spots of microbial activity in peat.

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Role of interacting soil engineers in the transfers of C-N-S in a

regenerating cutover peatland

Gicquel Aurélien, Binet Françoise, Briand Marie-Paule, Josseli Nathalie,

France André-Jean

UMR CNRS 6553 ECOBIO / University of Rennes 1, France

Soil organisms, because they change soil physical and chemical properties as well as microbial

communities, are efficient engineers that play a key role on the soil nutrient dynamics. Although the

coupling of carbon (C) and nitrogen (N) in soil have been well studied, such studies in peat soils are

scarce and even unavailable for sulphur (S). The aim of our study was to assess the impact of peat

bioturbation by an epi-anecic earthworm (Lumbricus rubellus L.) and/or a plant (Eriophorum

angustifolium L.) in the coupling of C-N-S in a regenerating cutover peatland, by using

simultaneously three isotopic tracers (13

C-15

N-34

S). We thus focused on C-N-S transfers in the

drilosphere and rhizosphere that are representative of the earthworm and/or plant activities. Labelled

organisms (three earthworms or two plants) were introduced in unlabelled peat mesocosm (2 litres).

Earthworm tissues, plants, representative peat samples of the drilosphere and the rhizosphere and

bulk peat were analyzed for their (‰)13

C, 15

N and 34

S by using an elemental analyser-isotope ratio

mass spectrometry (20-20 IRMS, Europa Scientific) after 3 and 18 days of incubation in a climate

chamber. We demonstrated rapid C, N and S enrichment of casts (x 1.3, x 70 and x 1.6 respectively)

and burrows (x 1.2, x 30 and x 1.2) derived from labelled earthworms and of rhizosphere (x 1.1, x 10

and x 1.1) derived from labelled plants compared to the bulk soil. With regard to Nitrogen, a

significant N transfer from labelled earthworms to unlabelled plants was shown, which reached up 4

% of 15

N (40 µg) within 18 days. Our study highlighted the key role of soil engineers in the cycling

of C-N-S in regenerating cutover peatlands by enhancing C-N-S transfers and providing nutrient

enriched peat hot spots that are expected to sustain higher microbial activities. Further studies will

therefore focus on microbial communities and C-N-S microbial uptake in the rhizosphere and the

drilosphere.

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Short-term effects of epigeic earthworms on the structure and activity of

microbial decomposer communities

Gómez Brandón Maria1, Lores Aguín Marta

2, Domínguez Jorge

1

1Universidad de Vigo

2Universidad de Santiago de Compostela

Epigeic earthworms live in organic horizons, in or near the surface litter, where they interact closely

with other biological components of the soil system, and in turn can affect the structure and function

of microfauna and microflora. In the present study we investigated the short-term effects of three

epigeic earthworm species (Eisenia andrei, Eisenia fetida and Perionyx excavatus) on the microbial

community structure and activity of three animal manures (cow, horse and rabbit), in comparison

with those in a control treatment. We evaluated the impact of these earthworm species on the

abundance of bacteria and fungi and their corresponding growth rates and on total microbial activity

(basal respiration) after one month. We performed a two-factor experiment with each combination of

manure and earthworm species. For this, plastic containers (2 L) were filled to three quarters of their

capacity with moistened and sieved vermiculite and inoculated with ten mature earthworms. Two

hundred grams (fresh weight) of manure was placed on a mesh (5 mm pore size) on the surface of

vermiculite. The containers were covered with perforate lids and stored at random in an incubation

chamber at 20 ºC and 90% relative humidity. We also included a control treatment that consisted of

100 g of manure incubated without earthworms. Each treatment was replicated three times. The

relatively rapid gut transit time of epigeic earthworm species resulted in the residues being

completely processed by the earthworms in one month. The discriminant analysis of the PLFA data

clearly distinguished between the animal manures after being processed by the three different epigeic

earthworm species, irrespective of what manure type was used. The separation between the

earthworm-processed manures and controls was also very clear. The earthworm species E. andrei

reduced in a large way the abundance of bacterial and fungal PLFAs relative to the control, although

this effect varied in function of the type of manure. The same pattern was observed with respect to

the bacterial growth rate and total microbial activity; however, no such changes were found in the

fungal growth rate as a result of earthworm activity. These findings provide a new insight into how

detritivorous earthworms affect the microbial ecology of the substrates that they inhabit and highlight

the potential of E. andrei in the biodegradation of organic residues.

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Effects of the transit through the gut of epigeic earthworms on the

structure and activity of microbial decomposer communities

Gómez Brandón Maria1, Aira Manuel

1, Lores Aguín Marta

2,

Domínguez Jorge1

1Universidad de Vigo

2Universidad de Santiago de Compostela

Earthworms can interact with the soil microbial community either directly or indirectly through their

feeding, burrowing and casting activities. Digestion of the ingested material, which occurs in a time

scale of hours, is the first step in this interaction process. Most of the current knowledge regarding

interactions between earthworms and microorganisms relates to soil-dwelling endogeic and anecic

species; as such, in the present study we evaluated the changes in the microbial community structure

(PLFA profiles) and activity (fluorescein acetate hydrolysis) of three animal manures (cow, horse and

pig) after passing through the gut of the epigeic earthworm species Eisenia andrei. Plastic containers

(250 mL) were filled to three quarters of their capacity with moistened and sieved vermiculite and

inoculated with twenty-five mature earthworms. One hundred grams (fresh weight) of manure was

placed on a mesh (5 mm pore size) on the surface of vermiculite. The containers were covered with

perforate lids and stored at random in an incubation chamber at 20 ºC and 90% relative humidity. We

also included a control treatment that consisted of 100 g each manure incubated without earthworms.

Each treatment was replicated five times. After three days, the earthworms were removed from the

containers, washed with sterile distilled water, and placed into a Petri dish to allow them to shed their

gut contents on moistened tissue paper for 24 h. After this time the gut contents from earthworms

from the same containers were pooled for analysis; the same amount from the control containers was

also collected. The passage of the three animal manures through the gut of the earthworm species E.

andrei led to a reduction in the viable microbial biomass measured as total PLFAs relative to the

control, irrespective of what manure type was used. A decrease in the abundance of bacterial and

fungal PLFA biomarkers was also observed as a result of gut transit, although this effect was more

pronounced in horse manure with respect to fungal populations. Moreover, we detected a reduction in

microbial activity relative to the control when E. andrei fed on cow and horse manure rather than pig

slurry. These findings reveal that epigeic earthworms play a key role in the decomposition of organic

matter via their intense interactions with microbial communities through the gut associated processes.

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Do earthworms prey on nematodes?

Laboratory food-preference experiment

Grabczysnka Olga, Makulec Grzegorz, Ilivea-Makulec Krassimira

Centre for Ecological Research of Polish Academy of Sciences, Poland

Soil nematodes and earthworms perform important functions in the soil system, but the relations

between these organisms are still unequivocal. In the presence of earthworms, nematode abundance

usually decreases. Common conclusion is that nematodes are digested by earthworms, but whether it

is accidental or selective preying is still ambiguous. Do earthworms choose nematodes selectively

and more often than the other available food types? The food preference of different species of

earthworms to different food types were studied in a laboratory experiment. The tested species of

earthworms were: Lumbricus terrestris, Lumbricus rubellus and Aporectodea caliginosa. As a food

source were chosen: bacteria cultured on agar, fungi cultured on agar, agar with a known number of

nematodes, agar with sterile oak-birch litter and as a control agar with fine-grained sand. One

specimen of earthworm was introduced to a centre of Petri dish (diameter 21 cm) with prepared food

sources. Cultures on agar were arranged near the circumference of a Petri dish, in equal distance from

the centre of the dish and other food types. Experiment was conducted for 72 hours. Presence of

animals on food during experiment was observed. The number of nematodes was evaluated after

termination of experiment. A methodology of this experiment, some difficulties connected with the

experimental design and observation procedure are discussed and preliminary results are presented.

The study was supported by the grant of Polish Ministry of Science and Higher Education (N N304

402038).

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Response of nematodes to earthworm abundance in the litter

Grabczynska Olga1, Ilieva-Makulec Krassimira

1, Uvarov Alexei

2

1Centre for Ecological Research of Polish Academy of Sciences, Dziekanów Lesny, Łomianki, Poland

2Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia

Nematodes are important component of soil food web. Presence of earthworms or changes in the

earthworm abundance affects abundance of nematodes. In a mesocosm experiment, the response of

nematodes in the gradient of earthworm population density in litter was investigated. Five-month

experiment was conducted in beech-oak forest. Each mesocosm was filled with 10L of soil and 100 g

of litter. Tested earthworms‘ species represented three ecological groups – epigeic (Dendrobaena

octadera, Lumbricus rubellus), endogeic (Allolobophora chlorotica, Aporrectodea caliginosa) and

anecic (Lumbricus terrestris). Each earthworm species was introduced to mesocosms in three

different densities – minimal, medium and maximal. Number of earthworms in each density level

varied between earthworms‘ species due to differences in the biomass. Results for litter may be

summarized as follow: 1) All investigated earthworm species reduced significantly the number of

nematodes, except D. octaedra. The most distinct effects were observed with maximal earthworms‘

density, which is related to increase of earthworm biomass in those treatments. The most severe

reduction in nematode abundance was observed in mesocosms with L. terrestris. 2) Considering the

ecological groups, epigeic and endogeic earthworms reduced nematodes number at similar level (on

the border of the significance). Impact of the anecic L. terrestris on nematode density was far

stronger in comparison to the earthworms‘ species from the other ecological groups. 3) In

mesocosms with high earthworms‘ biomass decrease in amount of litter was noted. In treatments

with low quantity of litter lower abundance of nematode was found. 4) At maximal density L.

rubbelus, an epigeic earthworm, reduced number of nematodes in litter only slightly stronger than A.

caliginosa, endogeic, soil dwelling earthworm. Impact of endogeic earthworms on nematodes

inhabiting litter should receive more attention. The study was supported by the grant of Polish

Ministry of Science and Higher Education (2P04F03030).

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Importance of ammonium content in the attraction or repulsion of

Collembola to the excreta of earthworms

Gutiérrez Mónica 1, Salmon S.

2, Fernández Rosa.

1, Trigo Dolores

1

1Universidad Complutense de Madrid

2Muséum National d´Histoire Naturelle

Previous studies have shown different reactions of several collembolan species to the excreta of

earthworms belonging to different families and ecological categories (Salmon 2001, 2004; Salmon

and Ponge, 2001; Salmon et al., 2005; Gutiérrez et al., in preparation). Trophic causes related with

the ammonium concentration were proposed. The objective of this study was to measure the

ammonium concentration in two types of excreta (casts and mucus-urine) of two earthworm species

in order to explain the contrasted response of collembolan species. The earthworm species used in the

experiments were the anecic Aporrectodea giardi (Ribaucourt, 1900) (Lumbricidae) and the endogeic

Hormogaster elisae Álvarez 1977 (Hormogastridae). The NH4+ concentration was determined in

fresh casts and in the mix of epidermial mucus and urine, following the Kjeldahl method (IPLA,

1984). To determine NH4+ concentration in mucus-urine, half-discs of filter paper impregnated with

the excreta of each earthworm species were analyzed. Earthworms had previously voided their

digestive tube by keeping them for 3 days in Petri dishes with moistened filter paper. They were then

placed in Petri dishes with six half disks of filter paper to saturate them with mucus-urine during 4

hours. NH4+ concentration was referred to fresh weight of mucus urine. The casts produced by

earthworms during these 3 days were collected daily and maintained at -20ºC until analysis. NH4+

concentration in casts was referred to dry weight of casts. Means of NH44+ concentration in casts and

mucus-urine of both earthworm species were compared by a t-test for independent samples. NH4+

content in casts was significantly ten times higher in A. giardi than in H. elisae. The rate of cast

production was 0.0755 g fresh body g-1

d-1

for H. elisae and 0.0425 g fresh body g-1

d-1

for A. giardi.

Conversely, the NH4+ content in mucus-urine was significantly ten times lower in A. giardi than in H.

elisae. The rate of mucus-urine production was 0.0007 g fresh body g-1

h-1

for H. elisae and 0.0015 g

fresh body g-1

h-1

for A. giardi. These results indicate differences in the nitrogen excretion way

between these anecic and endogeic earthworm species. Contrasted responses of collembolan species

to the excreta of earthworms (Gutiérrez et al., in preparation) may thus be attributed to different

nitrogen excretion way between earthworm species, and to variable sensitivity among collembolan

species to levels of ammonium concentration.

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Earthworm mating success: dependence on environmental conditions and

individual properties

Kati Saarinen

1, Ketoja Elise

1, Haimi Jari

2, Nederström-Alanko Aime

1, Nuutinen Visa

1

1MTT Agrifood Research Finland, FI-31600 Jokioinen, Finland and Department of Biological and

Environmental Science, FI-40014 University of Jyväskylä, Finland 2Department of Biological and Environmental Science, FI-40014 University of Jyväskylä, Finland

Reproductive success is an important element of individual fitness and mating frequency is one of its

key components. In earthworms the factors affecting mating success of individuals have remained

largely unexplored. The aim of this study was to find out how likelihood of mating in two earthworm

species with different ecology is related to i) environmental conditions, ii) population density, and iii)

the size of an individual. We utilized preserved earthworm samples that had been collected during

autumns of 2004 and 2005 from arable fields in 11 localities of Finland. The samples had been first

preserved in 1:1 mixture of formalin and ethanol and subsequently stored in 85% ethanol. The

presence of spermatophores on individuals was recorded and used as an indicator of recent mating

preceding the sampling. Spermatophores are formed on the body surface of the individuals during the

mating but in soil dwelling worms they detach in a few days. The study species were endogeic

Aporrectodea caliginosa Sav. and anecic Lumbricus terrestris L.. We used logistic regression to

model the probability of the occurrence of spermatophores. As explanatory variables we used i)

environmental conditions (rainfall, relative humidity and air temperature in different time periods

preceding the sampling and soil moisture during sampling), ii) number of adult individuals in the

sample and iii) weight of preserved individuals. Preliminary results show that the mating probability

of L. terrestris is positively related to the size of the individual and the cumulative rainfall during the

week before the sampling. We found no evidence for any relation between L. terrestris mating

success and population density, which ranged from 1 to 16 adults in the 0.25 m2 samples. High

likelihood of mating in large individuals may be due to their higher efficiency in mate searching

and/or assortative mating. The response to rainfall is likely to be explained by the mating behaviour

of the species: L. terrestris copulates on the soil surface and moist weather conditions provide

favourable conditions for surface activity. In A. caliginosa the statistical modelling is still in

progress, the likely presence of two subspecies in the material making the modelling more

challenging than in L. terrestris. We conclude that the present approach offers a useful tool for the

study of reproductive ecology of earthworms.

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Earthworm-mediated carbon and nitrogen losses via leaching and gaseous

emissions in riparian buffers

Kernecker Maria L., Whalen Joann

Department of Natural Resource Sciences, McGill University

Riparian zones occur at the transition between terrestrial and aquatic ecosystems, thereby regulating

nutrient flow across ecosystem boundaries. The hydrologic regime, vegetation and soil conditions in

riparian buffers may favour soil foodweb organisms like earthworms. It is well documented that

earthworms‘ feeding and burrowing activities stimulate carbon (C) and nitrogen (N) transformations,

leading to nutrient leaching and production of greenhouse gases (GHG) such as CO2and N2O. The

objective of our research was to identify how earthworms affect these processes in riparian buffers. A

literature review revealed that in agroecosystems, the aforementioned earthworm-mediated processes

have variable effects on nutrient leaching and GHG emissions depending on land-use practices.

There is limited information about how these processes are affected by the presence of earthworms in

riparian buffers. This led to a microcosm experiment on the effects of earthworms and riparian plant

litters on soil C and N cycling. Earthworms, soil and plant litter came from riparian buffers along the

Riviere-aux-Brochets, Quebec. The microcosms were made of PVC pipe (10cm dia. x 35cm tall),

hand packed with soil to a bulk density of 1.0 g cm-3 and wetted to 20% soil moisture content. The

experiment was a replicated (n=4) complete factorial design with twelve treatments comprising three

earthworm treatments (Lumbricus terrestris, Aporrectodea turgida, no earthworm) and four plant

litter treatments (soybean, switchgrass, deciduous tree, no litter). The 48 microcosms were kept in a

controlled temperature chamber at 12ºC. At days 1, 3, 5, 7, 10, 14 and then every two weeks during

the 20 wk experiment, soil in each microcosm was moistened with 150 mL water, cores were covered

with a vented lid equipped with a septa and incubated 24 h at 20ºC. Headspace air in each core was

analyzed for CO2and N2O, and soil leachate was analyzed for DOC and NO3-. A 10 g soil sample was

removed from each core and analyzed for NH4+, NO3

-and microbial N. At week 20, earthworms were

removed and soil was mixed with 1.6 g cm-3

Na-polytungstate to isolate the light fraction of organic

matter and assess the effect of treatments on C humification. Preliminary data indicate higher NO3-

leaching and CO2 production in microcosms with L. terrestris, compared to microcosms without

earthworms. Greater CO2 production was observed in microcosms with soybean litter, compared to

switchgrass or deciduous tree litters. These results highlight the important contributions of

earthworms and plant litter quality to soil C and N transformations in periodically flooded soils.

Results from this study will be linked with in-field measurements of earthworm demographics, soil

properties, and gas flux measurements to develop a landscape-level model of earthworm-mediated

nutrient cycling in riparian buffers.

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Microbial communities associated with three populations of Eisenia

earthworms - polyphasic approach

Koubová Anna, Pizl Vaclav, Chrozáková Alicia, Elhottová Dana

Institute of Soil Biology, Biology Centre ASCR, CZ-370 05, Ceské Budejovice, Czech Republic

The study was focused on the complex microbial changes during the passage of food material

through the gut of two earthworms, Eisenia fetida collected from forest soil and two populations of

Eisenia andrei originated in garden compost and large scale vermiculture plant, respectively.

Microbial community composition and biomass was determined in the gut and faeces of earthworms

as well as in feeding substrates. Phospholipid fatty acid analysis (PLFA) was used to assess total

microbial biomass, aerobes/anaerobes density and main community taxa composition. Bacterial and

Archaeal diversity was evaluated using polymerase chain reaction - denaturing gradient gel

electrophoresis (PCR-DGGE) and plate cultivation of bacteria completed with species screening of

dominant isolates (MIS Sherlock System). The passage of raw material (soil or organic compost)

through the intestine of all three earthworms under study resulted in a significant increase of total

microbial biomass and in enhanced density of anaerobes. Selective effect of the gut transit was

demonstrated by the increase of Gammaproteobacteria and microeukaryotes and the decrease of

Actinobacteria and Firmicutes in worm guts and faeces as compared with undigested food. The

diversity of cultivable bacteria decreased during the gut passage in all earthworms whereas the

bacterial counts were significantly (3 orders) higher in the gut and faeces of E. andrei than in those of

E. fetida. Shifts in the bacterial and Archaeal community composition were more apparent during the

passage of compost or vermicompost through the gut of E. andrei than during the passage of forest

soil through E. fetida. The polyphasic approach based on different views into the complex microbial

community added to the knowledge of the structural and diversity changes of the microflora during

the passage through the intestine of Eisenia earthworms.

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Earthworm-mycorrhiza interaction on growth of the Camedor palms

Lara Liliana1, Estrada-Segundo Margarita

1, Arcos-Solano José M.

1, Trejo Dora

1,

Barois Isabelle2, Ortiz Ceballos Angel I.

3

1Facultad de Ciencias Agrícolas, Universidad Veracruzana. Xalapa, Veracruz, Mexico

2Instituto de Ecología A.C.

3Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana. Xalapa,Veracruz, Mexico.

Management of the earthworms, arbuscular mycorrhiza fungus AMF) and plant roots are key

components of the below-ground agroecosystems, but their interacting effects on plant growth are

still poorly understood. An experiment was conducted in greenhouse to evaluate the influence of

presence or absence of native tropical earthworm Balanteodrilus pearsei on the development of AMF

on camedor palms (Chamaedora elegans) roots and the contribution of the symbiosis to palm

growth. When plants had the first leaf pair they were inoculated with AMF (Micofer-UV

consortium). The plants were harvested after 327 days (140 days after worms added) of growth. The

formation of external mycelium, vesicles and colonization were measured. The results suggested that

the earthworm activity significantly reduced AMF colonization and external mycelium. When

earthworms were added, the formation of vesicles and hyphae of AMF significantly increased.

However, the presence of earthworm had no effect on camedor palms root and shoot biomass. This

finding suggested that the earthworm activity affects AM root colonization by consuming soil,

building tunnels or feeding on extraradical mycelium.

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Interaction between earthworms (Eisenia fetida? and mycorrhiza (Glomus

intraradices) take positive effects on maize growth

Li Huan-Huan, Wang Chong, Yi Lou

College of Resources and Environmental Sciences, China Agricultural University

Arbuscular mycorrhiza fungi and earthworms are important components of the belowground part of

agricultural ecosystem. However, their interacting effects on crops growth are poorly understood. A

pot experiment was conducted to investigate the influence of epigeic earthworm Eisenia foetida,

Abuscular mycorrhizal fungi Glomous intraradices and their interactions on the growth and nutrition

uptake of maize. Arbuscular mycorrhizal fungi and earthworms significantly affected the

performance of maize, and these effects were not independent of each other. With the mycorrhiza or

earthworms lonely, the shoots biomass was significant higher compared with the control treatment.

Moreover, shoots biomass was higher when both AM and earthworms were present in the

microcosm. In the absence of earthworms the mycorrhizal association reduced the amount of N in

leaves, but when earthworms were present, this effect disappeared. It is possible that mobilized soil N

by earthworms reduced G. intraradices competed with the roots for available N. Available

phosphorus and ammonium-N in the soil were significantly lower when AM and earthworm were

added respectively. AM and earthworm had synergistic effect in the promoting soil organic carbon.

The formation of mycorrhizal structures in roots and hyphal length density in soil was measured at

harvest too. Earthworms caused an increasing in hyphe frequency and hyphal length density of AM

which might acquire more nutrition for mycorrhiza. Management of mycorrhiza-earthworm

interactions may be of value, particularly in low-input and organic agricultural systems, and deserves

further investigation.

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Interactions between invasive earthworms, arbuscular mycorrhizal fungi

and diazotrophic bacteria on native araucaria pine seedling performance

Martins Azevedo Trigo Priscila1, Dilmar Baretta

2, Brown George

3, Jurandy Bran Nogueira

Cardoso Elke1

1Escola Superior de Agricultura "Luiz de Queiroz" / Universidade de São Paulo (ESALQ/USP)

2Centro de Educação Superior do Oeste, Universidade do Estado de Santa Catarina

3Embrapa Florestas

The Araucaria angustifolia is a tree of high lumber and resin values, a source of food, and the most

important tree in the Mixed Subtropical Ombrophilous forest in Brazil, a rich ecosystem with many

endemic species. However, due to intense exploration for decades, the Araucaria is presently

considered a critically endangered species, and the understanding of this ecosystem is fundamental

for conservation and regeneration purposes. Soil organisms such as earthworms, arbuscular

mycorrhizal fungi (AMF) and diazotrophic bacteria play an essential role in nutrient cycling and can

increase plant performance. Many studies have evaluated the impact of each of these organisms

independently, but few have studied their interactions. Therefore, the aim of this study was to

evaluate the inoculation effects of invasive earthworms (Amynthas corticis), AMF (Gigaspora rosea)

and an isolated of diazotrophic bacteria, and the interactions between each of these, on the

performance of A. angustifolia seedlings. The plants were grown in greenhouse conditions in a block-

type randomized design with a factorial layout of 2x2x2, being: presence and absence of earthworms

(A. corticis), presence and absence of AMF (G. rosea) and presence and absence of diazotrophic

bacteria (isolated S32274), in eight replicates, evaluated at 150 and 250 d after transplanting. The

following parameters were evaluated: plant height and diameter, shoot and root biomass, shoot

nutrient content (P, N), number of AMF spores, root mycorrhizal colonization rate and earthworm

survival, reproduction and fresh biomass. Plant biomass and shoot nutrient content were affected by

the three-way interaction of earthworms, AMF and bacteria. AMF inoculation enhanced A.

angustifolia performance, while bacterial inoculation alone had no significant effect on plants, and

earthworms had low survival rates and tended to reduce plant biomass. Microbial-faunal interactions

can lead to important changes in plant growth and should be considered in future research and

development of sustainable Araucaria forest management practices.

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Species segregation and distance-related decline in the similarity of

earthworm communities

Monroy Fernando, Aira Manuel, Domínguez Jorge

Universidad de Vigo

Earthworms have a major impact on soil structure and nutrient dynamics. The type and intensity of

changes caused by these soil invertebrates depend on their ecological category —epigeic, endogeic,

anecic— and therefore on their community composition. Although earthworm communities differ

between sites, little is known about the factors controlling the spatial distribution of earthworms and

the structure of their communities. We used data from 107 locations in northwestern Spain to

investigate the degree of similarity of earthworm communities over distances ranging between 10

and 200 km. The locations were exhaustively sampled using the formalin – hand sorting method and

the collected earthworms were identified at the species level. Community similarity indexes were

calculated using pair-wise comparisons between locations. The correlation between these indexes and

geographic distance was calculated using a Mantel test. We found a significant decline in community

similarity with distance. This decline was not associated with distance-related gradients in soil pH

and organic carbon, suggesting that factors other than soil chemistry, e.g. dispersal limitations or

competitive interactions, may be involved in the decline of community similarity. We examined the

co-occurrence patterns of earthworm species in our sampling region and the results showed that some

species were spatially segregated. These results support the idea that species interaction processes

were also shaping earthworm communities. Overall, we found that geographic distance is a

significant factor accounting for differences in earthworm community composition, and that the

observed decline in similarity with distance may be related to differences in dispersal abilities and to

competitive interactions between earthworm species.

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The midden sites of Lumbricus terrestris L. as a source of greenhouse gas

emissions in arable no-till soil

Nieminen Mervi1, Regina Cristiina

1, Hurme Timo

1, Mikola Juha

2, Nuutinen Visa

1

1MTT Agrifood Research Finland, FIN-31600 Jokioinen, Finland

2Department of Ecological and Environmental Sciences, University of Helsinki, FIN-15140

Due to enhanced carbon sequestration, no-till is regarded as a useful method for reducing carbon

dioxide (CO2) emissions from arable lands. However, no-till has also been shown to elevate nitrous

oxide (N2O) emissions in some soils, which could offset the atmospheric benefits of no-till. The role

of earthworms in gas emissions from arable soils has recently received interest and earthworm

activity has been shown to contribute for instance to N2O emissions. No-till often leads to increased

population densities of the large, deep burrowing earthworm Lumbricus terrestris L. We therefore

established a field and a laboratory experiment to find out if the presence of L. terrestris can

significantly affect the emissions of the greenhouse gases N2O, CO2 and CH4 from arable no-till soil.

In the long-term no-till field in S-W Finland, gas emissions were measured five times in two weeks

on L. terrestris midden and adjacent non-midden sites (n=10) in October 2008 using plastic chambers

(diameter=15 cm). The laboratory experiment (duration 15 weeks) was established in November

2008, with the soil, L. terrestris individuals and straw collected from the same no-till field. The soil

was sieved and packed to PVC-tubes (diameter 15 cm, height 45 cm), and one large L. terrestris

individual was placed to half of the tubes, the tubes without a worm serving as controls (n=15). Gas

emissions were measured 12 times in approximately one week intervals. Although statistical analyses

are still underway, preliminary results from the field experiment suggest that N2O and CO2 emissions

were significantly higher on L. terrestris midden than on adjacent non-midden sites. Methane (CH4)

emissions differed only at one measurement, and then the non-midden sites emitted more CH4 than

the midden sites. At the last measurement, when the gas emissions were measured with and without

middens or residues in the sampling area, L. terrestris sites were found to emit more N2O, CO2 and

CH4 than the non-midden sites both before and after the removal of middens / residues. There was

even an indication of N2O emissions at L. terrestris sites being higher after than before the removal

of middens. In the poster, we will present a comprehensive summary of the results of the field and

the laboratory experiment. We will also try to draw the first conclusions of the net impact of L.

terrestris activity on greenhouse gas emissions from no-till soils.

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Effect of the moisture of soil in position of cocoons of earthworms

Pontoscolex corethrurus (Annelida; Oligochaeta, Glossoscolecidae)

Ortíz Ceballos Angel I. 1, Sánchez-Cortéz Mayra T

1, Brown George

2

1Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana

2EMBRAPA, Brasil

The ability of Pontoscolex corethrurus to colonize an ample variety of agroecosystems is well-

known and to understand the factors that take part are of great relevance to design new practices that

include their handling, to improve soil fertility. The present study had as objective to investigate if

the earthworm P. corethrurus has a preference for certain soil moisture to deposit its cocoons and

their influence in the incubation period. The study was divided in two stages. First, in terrariums

modified (0.35 x 0.45 x 0.005m) with an adult earthworm (clitellum), manipulating (50 days) in a

combined form, the moisture content (25, 33 and 42%; with 5 replicates each) in three depths of

ground. Second, in order to eliminate the possible effects of the interaction of three moist within the

terrariums, manipulating (60 days) in separated from the soil moisture content (25, 33 and 42%, with

5 replicates each) registering the number, position and the incubation duration of cocoons deposited

by P. corethrurus. The preliminary results indicate that the humidity of soil, independent of the

depth, had a significant effect in the cocoon position. P. corethrurus deposited cocoons in the three

moist of the soil; nevertheless, the major and minor numbers of cocoons were deposited with a

humidity of 33 and 42%, respectively. In addition, the youthful earthworms emerged in minor and

major time with an intermediate and high moist, respectively.

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Two parasitic mites in the cocoons of the pantropical earthworm

Pontoscolex corethrurus (Annelida: Oligochaeta, Glossoscolecidae)

Pascacio Velázquez José M. 1, Ortiz Ceballos Angel I.

1, Palacios Vargas José G.

2

1Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana

2Laboratorio de Ecología y Sistemática de Microartrópodos, Departamento de Ecología y Recursos Naturales.

Facultad de Ciencias, UNAM, Mexico

It is well documented that some mites parasitize cocoons of the European lumbricid species. In a

previous study, damage was observed in developing cocoons of the tropical earthworm Pontoscolex

corethrurus associated with the presence of mites. In this study we assessed whether the parasitic

mites the cocoons of the P. corethrurus. In the garden of INBIOTECA for a period of 70 days (from

September to December 2008) at a distance of 100 m, each week (10 times) 20 cocoons were

collected randomly by manually (depth of 0-20 cm). In the laboratory, the cocoons were individually

placed in Petri dishes with filter paper and deionized water under 23± 5 ºC. During the incubation

was recorded the number of earthworm hatched, number of cocoons parasitized and taxonomic

identification of species mites that feed on the cocoons incubated. The results showed that 12% of

cocoons were parasitized by mites. Two species of mites were identified belonging to the families

Anoetidae (genus Histiostoma) and Glycyphagidae. It is concluded that mites are natural enemies to

parasitic cocoons of P. corethrurus. Future studies should determine whether the distribution of P.

corethrurus is limited by the geographical distribution of mites and if they also parasitized cocoons

of the other tropical species (exotic and native).

197


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Impact of anthropic pressure gradient on earthworms in saline soil in

China forest. Consequences on ecosystems services as soil structuration

and hydraulic conductivity

Pérès Guénola1, Roualt J.

1, Piron Denis

2, Li Yang

2, Liu C.

2, Hallaire Vincent

3, Cluzeau

Daniel1

1University Rennes 1, UMR CNRS "Ecobio" 6553, Station Biologique, 35380 Paimpont, France

2School of

Agriculture and Biology . Shanghai Jiaotong University . 800,Dongchuan Road. Shanghai, 200240. P.R.China

3INRA, UMR Sol Agronomie Spatialisation, Agrocampus-INRA Rennes 65 Route de Saint Brieuc CS 84215

35042 Rennes Cedex. France.

Since two years ago, China has noted the increase of environmental problems as the same time as an

increase of economic and demographic growth. Chinese government decided to take some measures

to resolve environmental problems. Thus, it‘s now a challenge to fight against environmental

problems as decline in biodiversity, soil pollution or soil erosion, and thus many local programs as

those proposed in Shanghai was developed in order to find solutions. This study is part of a French-

chinese cooperation, the ―ARCUS‖ Project, which is an ambitious program from Shanghai

municipality for Chongming Island. The objective is to create the first eco-island with sustainable

development in terms of eco-agriculture, water management, waste recycling, renewable energy and

eco-tourism. This island should be used as a model for Chinese town management in the future. The

first goal of this study is to assess the impact on earthworm communities of an anthropic pressure

gradient under forest and cultivated fields in a saline soil; earthworms are studied through their

bioindicators state. The second goal is to assess the consequence on ecosystem services as soil

structuration (bioturbations) and soil hydraulic conductivity (Kh). The originality of this study was to

study both biological and physical at the same time and the same place. Four treatments were

compared (forests of Metasequoia, 8 years old, 15 years old, 30 years old and a wheat field, 400 m²

each). Earthworms were sampled using formaldehyde extraction combined to hand sorting. Soil

structuration was mapped and quantified applying a new field methodology based on the description

of bioturbations created by earthworms and vegetation. Conductivity was measured using

infiltrometers at 3 different water potentials. Results showed low earthworm species richness in all

treatments, explained by salinization combined to anthropic actions, especially under wheat treatment

leaded in the erosion of earthworm density. The quantity and quality of bioturbations observed at the

40th first centimeters of soil appeared strongly influenced by anthropic gradients. Soil water

conductivity (Kh) in wheat was very low (impermeable soil) while it was strongly higher under forest

due to important macroporosity; biological activity (macrofauna and roots) was probably the main

factor that explained this structuration of soil forest. All these investigations established an

interesting perspective of tool transfer towards stakeholders.

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The effects of earthworm addition on nitrous oxide and carbon dioxide

fluxes in clover and grass enclosures

Prieto Roberto, Whalen Joann

McGill University, Canada

Earthworms are the dominant macrofauna in perennial forage systems. They profoundly impact soil

in situ conditions and consequently microbially-mediated processes. The earthworm gut and

structures (casts, burrows and middens) are proposed as hotspots of carbon dioxide (CO2) and nitrous

oxide (N2O) production, mostly based on laboratory studies in the absence of plants. A field study

was conducted to determine how increasing the population of two earthworm species would affect

the CO2 and N2O flux from red clover (Trifolium pratense) and grass (Elytrigia repens) forage

systems. The experimental site was in Ste. Anne de Bellevue, Quebec, Canada where adjacent red

clover and grass systems were found on a Chateauguay clay loam (pH = 6.5, 78.12 g organic C kg-1

).

Enclosures were 1 m2 soil blocks surrounded by polyethylene plastic to a depth of 30 cm. Earthworm

species added were the anecic Lumbricus terrestris L. or the endogeic Aporrectodea turgida (Eisen).

The experimental design was a factorial arrangement of earthworm species (anecic alone, endogeic

alone, anecic plus endogeic) and population levels (250, 500 and 1000 earthworms m-2

) plus a control

with no earthworm addition. These 10 treatments were randomly assigned and replicated in 4 blocks

in each forage system, for a total of 80 experimental enclosures. Gas samples were collected in the

fall of 2009 using a vented closed chamber system, the CO2 and N2O concentrations were determined

with gas chromatography and gas fluxes (mg m-2

h-1

) were calculated. Grass enclosures with added L.

terrestris had significantly (P<0.05) greater CO2 fluxes than with A. turgida or mixed species.

Earthworm species did not affect the CO2 flux in the clover system, nor did the population level. The

CO2 flux was up to 2.5 times greater from grass than clover enclosures, which we attributed to the

denser and more extensive rhizosphere under grass that promoted soil respiration. The N2O flux in

the clover enclosures increased linearly (R2 = 0.993) as the number of added earthworms increased

(0, 250, 500 and 1000 m-2

); thus, earthworms stimulated N2O emission in the clover system. No

effect of earthworms was observed in the grass enclosures. Moreover, N2O flux was up to 11 times

greater in clover than in grass, likely due to greater mineral nitrogen availability from decomposing

clover residues. In conclusion, increasing earthworm populations had little effect on CO2 flux but

stimulated N2O flux from a perennial clover system.

199


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Direct and indirect effects of deer on the native earthworm, Eisenoides

carolinensis

Ransom Tami1, Rearick Daniel

2, Kintz Laura

3, Burke Katherine L.

1

1University of Virginia

2Clarion University

3Mountain Lake Biological Station

Invasive earthworms are known to benefit from the high quality organic matter provided by deer

fecal pellets, but little is known about the effects of deer on native earthworms in the Southeastern

United States. We proposed that native earthworms would also benefit from increased organic matter

provided by deer fecal material. We examined potential interactions between a common aboveground

herbivore, the white-tailed deer (Odocoileus virginianus), and earthworms using a laboratory

experiment and field exclosures. For the laboratory experiment, our hypothesis was that a native

earthworm, Eisenoides carolinensis, would consume deer fecal pellets at a similar rate to an invasive

earthworm, Lumbricus terrestris. Both species fared better in treatments with deer pellets alone and

with deer pellets plus leaf litter in relation to the treatment with leaf litter alone. For the field

component of our study, we set up 18 field plots: exclosures, controls, and high deer fecal density

plots; deer exclosures were constructed in 2006. Octet electroshocking was used to evaluate the

densities and total mass of earthworm populations relative to deer fecal pellet densities. Deer were

found to negatively influence large juvenile E. carolinensis in mass and number (no invasive

earthworms were found on the field plots). The field and laboratory portions of our study appeared to

give contradictory results: in the field, earthworm densities were negatively related to deer fecal

pellet counts, but native earthworms gained more mass in the presence of fecal pellets (compared

with leaf litter) in the laboratory. However, we also measured soil and vegetation characteristics on

all field plots and used path analyses to explore how deer might negatively influence native

earthworms. The results of our path analyses and model selection support the hypothesis that deer

negatively influence E. carolinensis via indirect effects on understory plants. This study highlights

the importance of above- and below-ground interactions in forest ecosystems.

200


5th to 10



Effect of the intestinal transit and mucus of the endogeic earthworm

Pontoscolex corethrurus on bacterial communities from two tropical soils

Roy Virginie1, Barois Isabelle

2, Miambi Edouard

1, Giusti-Miller Stéphannie

1, Bello Julio

2,

Lavelle Patrick3, Mora Philippe

1

1Université Paris-Est Val de Marne

2Instituto de Ecologia A.C.

3Institut de Recherche pour le Développement

Endogeic earthworms are the most active ecosystem engineers in most tropical soils. Their energy

and nutrient needs are thought to derive from a mutualistic relationship with ingested soil

microorganisms. This assumption is supported by the observation of significant changes in microbial

activity during gut transit and in casts of different ages. For the first time, changes in bacterial

communities during transit through the gut of a tropical endogeic earthworm, Pontoscolex

corethrurus, were analyzed by PCR-DGGE fingerprinting. The bacterial community in the gut

content was different from that in the soil, demonstrating a selection effect. The native soil of the

earthworm was shown to have a significant effect on the structure of the ingested bacterial population

and no gut-specific taxa were observed. In vitro incubations of soil with added extracted intestinal

mucus were compared with incubations with added water or glucose instead of mucus. The mucus

had a specific effect on the composition of the bacterial communities.

201


5th to 10



Earthworms Ecology in the Soil of Estação Experimental Cascata,

Embrapa Clima Temperado, Pelotas, RS, Brazil

Schiavon Greice de Almeida1, Schiedeck Gustavo

2, De lima Ana Claudia Rodrigues

1,

Schwengber José Ernani2, Jahnke Dênnis Silveira

2

1Universidade Federal de Pelotas Pós Graduação em Sistemas de Produção Agrícola Familiar;

2Embrapa Clima

Temperado, Estação Experimental Cascata.

In the world, earthworm diversity is estimated to approximately 5900 species. However, little is

known about the diversity of earthworms in Brazil. The present study had the objective to estimate

the population density and diversity of earthworms in the Estação Experimental Cascata (EEC),

Pelotas, RS, Brazil. The study was realized in the EEC, the site has an area of 150 ha, with the

definition of seventeen sampling sites, from an area image with their geographical coordinates. After

the characterization of the site, the points were grouped in ecosystems: plantation area, secondary

forest, riparian forest, plantation area of exotic trees and area of initial successional stages. The

samples were realized in the spring of 2008 and in the summer of 2009, with two complementary

methods of collection; each sites had a collect area of 0,4375 m2. The soils were taken to the

laboratory, in which the soil was made manual sorting. Then, the earthworm specimens were

preserved, counted and identified. Each treatment had four replicates, with the exception of AE and

SI. For data analysis, principal component analysis (PCA) was used. Three earthworm families were

found: Glossoscolecidae, Megascolecidae and Acanthodrilidae. In the spring collection five

earthworm species were identified: Glossoscolex sp.1, Glossoscolex sp.2, Urobenus brasiliensis,

Amynthas gracilis, Microscolex dubius and juvenile unidentified earthworms. In the sample of

summer four earthworm species were found: Glossoscolex sp.1, Glossoscolex sp.3, Urobenus

brasiliensis, Amynthas gracilis, one unidentified species and juvenile earthworms. The main

correlation was between the plantation areas and the specie Amynthas gracilis. This demonstrates that

the inadequate management in agroecosystems can increase the population density of exotic

earthworm species.

202


5th to 10



Ecology of Soil Macrofauna of Estação Experimental Cascata, Embrapa

Clima Temperado, Pelotas, RS, Brazil

Schiavon Greice de Almeida1, Schiedeck Gustavo

2, De lima Ana Claudia Rodrigues

1,

Schwengber José Ernani2, Jahnke Dênnis Silveira

2

1Universidade Federal de Pelotas Pós Graduação em Sistemas de Produção Agrícola Familiar

2Embrapa Clima Temperado, Estação Experimental Cascata.

The soil is an habitat for a wide variety of organisms, mainly, invertebrates that act in different ways

in this environment. The soil invertebrates are classified as: microfauna, mesofauna and macrofauna.

This study had the objective to evaluate the density, biomass and diversity of groups from the soil

macrofauna, in the area of Estação Experimental Cascata (EEC), Embrapa Clima Temperado,

Pelotas-RS. In order to understand the ecological processes in natural, anthropogenic ecosystems and

in the agroecosystems. The study area has about 150 ha. The sampling points were defined without

choosing specific areas, using an overlapping grid of points, 300 m distant from each other, in the air

image of the EEC. After the points were located in the field, from the pre-defined geographic

coordinates, using GPS and grouped by ecosystems. The ecosystems found were planting area,

secondary forest, riparian forest, plantation area of exotic trees and area in the initial stage of

succession. Samples were collected in the spring 2008. For the survey of macrofauna, two methods

of collecting were used: the method TSBF (Tropical Soil, Biology and Fertility) and extracting

solution of formaldehyde to 0.5%. The planting areas and the areas in initial successional stage

comprised 13 taxonomic groups. However, secondary forest, the riparian area and the planting of

exotic trees had 15 taxonomic groups. In plantation areas, the most representative taxonomic groups

were Hymenoptera, Isopoda and Isoptera. In the remaining forest and the area in initial stage

successional, the groups with higher density were Hymenoptera, Isopoda and Isoptera. In the area of

riparian forest, the most representative groups were Isoptera, Hymenoptera, Chilopoda and

Oligochaeta. The plantation area of exotic trees was represented by groups Hymenoptera,

Amphypoda, Isopoda and Isoptera. Therefore, this work emphasize the importance of the orders

Hymenoptera, Isopoda and Isoptera.

203


5th to 10



Comparative ecology of earthworm population between natural and

manmade conditions

Sun Zhenjun1, Fang Weilin

2, Qiao Yuhui

1


2Bocom Pharmaceuticals Ltd.

In the natural ecosystem, the species composition, abundance and biomass of earthworm population,

apparently were influenced by vegetation and soil types, and varied essentially in relation to soil

organic matter content and anthropocentric actions, and soil temperature, moisture and pH value.

Some optimum ecological factors for worm growth are not best for worm reproduction, for instance,

temperature 25oC, moisture 70% and pH 6 are most suitable for E. fetida to cocoon production,

while temperature 18oC, substrate moisture 65% and pH 8-9 are most suitable to increase body

weight. In cultivated fields, most earthworms live within a 20 cm depth tillage layer due to the

presence of rich nutritional elements and favourable pH value; whereas in artificial substrate E. fetida

can descend to the 70 cm depth, or even deeper, owing to even-distributed nutrients and uniform feed

texture. But 99.2% of earthworms dwelled within the first 50 cm depth of substrate. It was suggested

that the substrate layer could be extended to a 50 cm depth. Vertical distribution of earthworms was

related to population density. In fact, earthworms mainly concentrated on the upper soil layer,

whereas in substrate, when the spatial competition between worm individuals intensifies with

population density increase, worms tend to go down to lower substrate layers. There may exist a

lower threshold density of ca. 60,000 ±10,000 ind.m2 , over which earthworm migration would occur.

Whether in natural soils or in substrate, most earthworm cocoons were deposited in the upper 15 cm

layer; this characteristic possibly evolutes over a long period of time. Worms, however, dwelled in

deeper layer in most time, which may be termed "non-uniformity of egg-laying and living in vertical

space". A depth of 15-20 cm may be sufficient for breeding worms. Under those experimental

conditions the clitella of E. fetida appeared 60-70 days after cocoon appearance. The maximum

individual weight was reached between 90-100 days. The worm density was not relative to the time

in which earthworms start to breed or individuals were harvested. E. fetida is a species of higher

reproduction rate and fast-growth. It may be more favourable to rear cocoon-laying and body-

fattening worms in different worm beds on the basis of the characteristics of E. fetida mentioned

above. On the base of substrate preference and vermiculture needs, the earthworms can be grouped in

compost-preferring species and soil-preferring species.

204


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Session 6

Earthworms and Soil Fertility

205


5th to 10


Plenary lecture – Session 6

Soil improvement and earthworms: Production and behavioural results

since ISEE4

Butt Kevin R.

University of Central Lancashire

Over the past twenty years a host of experiments have been conducted in these research areas. A

personal view is presented to demonstrate developments which may assist promotion of soil fertility

in given soils.

206


5th to 10


Oral – Session 6

Earthworms as new pests of irrigated rice in Brazil

Freitas Barrigossi José Alexandre1, Pedretti Júnior Carlos

1, Bartz Marie Luise Carolina

2,

Lima Ana Claudia R.3, Gassen Dirceu N.

4, Pasini Amarildo

2, Brown George

5

1Embrapa Arroz e Feijão, Brazil.

2Departamento de Agronomia, Universidade Estadual de Londrina, Brazil

3Federal University of Pelotas, Campus Universitário, Brazil

4Cooplantio, Brazil

5Embrapa Florestas, Brazil.

Earthworms have been reported as pests of rice in various countries, mainly in SE Asia and Australia,

but only recently have they been accused of damage to rice cultivation in Brazil. Nevertheless, few

studies have been performed on earthworm communities in rice fields in Brazil and little is known of

the ecology of the species encountered thus far. Therefore, the present study was undertaken to: a)

evaluate the species associated with rice damage in three sites, and b) assess the relationship between

earthworm populations and rice yields at two sites. Earthworms were collected in Miranda and Rio

Brilhante, Mato Grosso do Sul and in Santa Maria, Rio Grande do Sul. At the two former sites, rice

production was also assessed in a 1m2 area, and in its center, earthworm abundance assessed using

the TSBF method (25 x 25 cm monoliths). The relationship between rice yields and earthworm

abundance was assessed and correlation coefficients tested using Student‘s T-test. In Santa Maria, the

species associated with yield losses was a small, thin Ocnerodrilid, Eukerria eiseniana. In Miranda, a

new species of earthworm in the Criodrilidae family was found, and the relationships between

abundance of this species and rice grain yields (r =0,35; P=0,13) or number of stalks (r= -0,08;

P=0,71) were not significant. In Rio Brilhante, two species predominated, a Eukerria sp., and a

moderate-sized Glossoscolecid (Anteoides sp.). The relationship between earthworm abundance and

rice grain yields or number of stalks was also non-significant. Therefore, earthworm abundance does

not appear to be directly negatively related to rice production. Nevertheless, yield losses may result

from high infestation due to plant lodging, which impedes adequate machine-harvesting of the rice

grains. Further research is needed to evaluate the extent of the problem in rice fields throughout

Brazil, and find environmentally-friendly means of reducing potential damage.*presenting author

207


5th to 10


Oral – Session 6

Relationship analysis between earthworm population and soil fertility in

different land-use types

Liao Yan, Zhang Ning, Wang Chong, Sun Zhenjun

Agriculture University China

Soil biological fertility was influenced earthworms and land use. A field survey was carried out in

Quzhou County, Hebei Province of China, for studying relationship between earthworm population

and soil fertility in different land-use types. In the survey we investigated earthworm population

characteristics, including species abundance, population density, biomass, age-structure and H‘index,

which can indicate the biodiversity pattern of earthworm--were employed to investigate the effects of

different Land--use types on earthworm biodiversity pattern. The relationship between Land--use

types and soil character was discussed after measuring soil character under ways of soil using. The

results indicated that: The number of earthworm species, its population density, biomass, and age

structure and diversity index were obviously related to most indices of soil chemistry and biological

characteristics, also responding to soil fertility in different land use. With earthworm population

characteristics as the evaluation index, the result corresponded with that of index evaluation using

soil biological characteristics and soil chemistry. Therefore, it is practical to synthetically appraise

soil biological fertility using earthworm population characteristics.

208


5th to 10


Oral – Session 6

Effects of mouldboard ploughing on changes with time in earthworm

abundance, in an organic farming system in Northern France

De Oliveira Tatiana

1, Bertrand Michel

1, Roger-Estrade Jean

2 1UMR 211 Agronomie, INRA AgroParisTech, France

2AgroParisTech, département SIAFEE, France

In cultivated fields, earthworms are soil fertility major actors. Adopting respectful agricultural

practices is necessary to protect, promote and optimize their activities. Mouldboard ploughing is

often recognised to have a greatest effect on earthworm abundance and activity. However, this real

impact is not well known and seems to be very variable as a function of the earthworm ecological

type. Moreover, the mechanisms are still debated: direct mortality, food resource decrease,

reproduction capacity reduction or habitat destruction? Literature gives contradictory answers to

those questions. To contribute to this issue, we set up a field trial in which changes in earthworm

abundance and community composition were monitored during two month after ploughing

(November to December 2009). The experiment was located in Northern France (49°7'N, 1°42'E) on

an organic field converted since 2001. After a chisel plough operation over the whole surface,

ploughing was performed on one part of the field (ploughed Pl) the other remaining unploughed (N-

Pl). Barley was sown the same day (12th

of November). Initial population was counted the day before

ploughing, and then evaluated 1, 14, 35 and 55 days after ploughing. Ten samples were taken in the

Pl zone and ten in the part left N-Pl. The community structure exhibited a strong dominancy of the

endogeics (anecics <10%). In the N-Pl area, earthworm abundance followed a positive trend,

showing a 20% increase after two months of monitoring. At the opposite, in the Pl area, the initial

density was, after 55 days, nearly divided by two. During the first two weeks, no difference appeared

between the Pl and N-Pl areas. Ploughing response was delayed. Taking the age classes, ploughing

was detrimental either for the juveniles and the adults. Concerning the species, results show

surprisingly a difference between the two most abundant endogeic species: Apporectodea caliginosa

were dramatically reduced while Allolobophora rosea exhibited the same evolution in the Pl and N-

Pl areas. We hypothesized that: 1) ploughing direct mortality was, in this experiment, negligible.

However, no birds (e.g. common gulls) followed the tractor during the operation and 2) ploughing

had a detrimental effect on survival or reproductive capacity, for example by decreasing the viable

cocoon stock, exposing them to desiccation and predation. Taking the species results, further

investigations will be needed to understand the different responses of the endogeics.

209


5th to 10


Oral – Session 6

Effect of management practices and earthworm biostructures on soil

hydraulic conductivity

Bottinelli Nicolas1, 2

, Hallaire Vincent1, 2

, Menasseri-Aubry S.1, 2, 3

, Le Guillou C.1, 2

,

Benard Y.1, 2

, Cluzeau Daniel4

1INRA, France

2Agrocampus Ouest, France

3Université européenne de Bretagne, France

4Université Rennes 1, France

This study aimed to evaluate the effect of management practices on soil hydraulic conductivity (Kh)

by studying earthworm biostructures. Two types of tillage were compared (moldboard plowing: MP

and no-tillage: NT) and two types of fertilization (mineral and poultry manure), seven years after

their establishments. Measurements were carried out under wheat crop during four sampling periods

at 2- to 7-cm depth. We measured on soil thin sections (i) three indicators of earthworm activity: the

area of individual casts, coalescent casts and burrows and (ii) soil macroporosity. The Kh (at -0.05

kPa) was measured at the same times and in the same locations than soil macroporosity. At all four

sampling dates, the soil macroporosity and Kh were the lowest under NT while earthworm activity

was the highest. Poultry manure, which was applied at agronomic rates, affected neither the

macroporosity nor Kh nor the amount of earthworm casts but increased slightly burrows abundance.

Regardless of the tillage treatments, Kh increased in spring and decreased in summer. Part of this

variation was attributed to rainfall events and earthworm activity which acted on the macroporosity

(shape and size) under MP and on the pore connectivity under NT. This study highlighted: (i) that

earthworm activity is not capable to replace the effects of the mouldboard ploughing even after seven

years of no-tillage under poultry manure and (ii) the type of tillage determines how the earthworm

activity promotes Kh.

210


5th to 10


Oral – Session 6

Evidence of the involvement of the auxin pathway in the impact of

earthworm on plants

Puga-Freitas Ruben, Blouin Manuel

Université Paris-Est Créteil

Earthworms are susceptible to affect plant growth through a variety of mechanisms such as trophic

relationships, physical engineering, modifications of microbial community or the emission of signal

molecules. The relative importance of these different mechanisms has not yet been elucidated.

Extracts from earthworm‘s casts have been shown to produces auxin-like effect on plants. Here, we

investigated the hypothesis that earthworm effect on plant growth was due to these auxin-like

molecules. In a laboratory experiment in microcosms, we compare the effect of earthworm presence

either on a wild plant genotype or on a mutant plant genotype with a deficient gene implicated in the

auxin pathway. We choose the Arabidospis thaliana double mutant aux1-7;axr4-2, which has a

strongly altered auxin transport. This results in a reduced sensitivity to auxin which in turn induce a

dwarf phenotype. As earthworms are susceptible to produce auxin-like compounds, they should by

this way compensate the deficient auxin transport towards the roots and revert the mutant phenotype

to the wild one. Earthworm presence induced a negative effect on Arabidopsis thaliana wild type

(shoot biomass decreased by 39%), this negative effect on the wild type was probably due to an

inhibitory effect of high auxin concentration but a huge positive effect on the aux1-7;axr4.2

Arabidopsis thaliana double mutant with an impaired auxin transport (shoot biomass increased by

780%). The positive effect on the mutant was likely due to the compensatory effect of earthworms on

auxin concentration in roots, as stated in the hypothesis. Root length and root surface area of the wild

type decreased by 55% and 51% respectively in the presence of earthworm. For the mutant, root

length and root surface area of increased by 474% and 550% respectively in their presence, for a final

surface uptake equivalent to that of the wild type in earthworm presence. This absence of difference

between root system of the mutant and wild type suggested that root development, which is under

hormonal control, was at the origin of the phenotype reversion in the presence of earthworms. Our

results validated the idea that signal molecules emitted from earthworm casts can affect plant growth.

This stresses on the importance of signals as an important but neglected mechanism as compared

with trophic relationships and physical engineering.

211


5th to 10


Oral – Session 6

Darwin’s dream comes true: NIRS allows quantifying the impact of

earthworms on soil and Carbon dynamic

Jouquet Pascal1, Henry-des-Tureaux Thierry

1, Brunet Didier

1, Tran Duc Toan

2

1IRD, France

2 SFRI

Far less famous than his work on Natural Selection, Darwin was also a great soil ecologist who

stressed the importance of earthworms in soil and landscape evolution. To date, however, we are still

unable to precisely quantify the impact of earthworms on soil processes. In this communication, we

review several studies that aimed to determine the specific signature of cast aggregates and to

quantify the importance earthworms play on soil erosion. Soil erosion was studied in different

sloping lands in Vietnam and Near Infrared Reflectance Spectroscopy (NIRS) was used to generate

signatures corresponding to different soil surface aggregates (aboveground soil casts produced by

earthworms vs. surrounding topsoil). The NIRS-signature of eroded soil sediments was compared to

those of casts and of the surrounding soils. Our results confirm that NIRS spectra might be used as

―fingerprints‖ to identify the origin of soil aggregates. Although earthworms are assumed to play a

favourable role in promoting soil fertility and ecosystem services, our results showed that sediments

might be almost entirely constituted of cast aggregates. Two hundred years after Darwin‘s birth, we

now have of a relevant method to quantify the true impact of earthworms on landscape evolution.

212


5th to 10


Oral – Session 6

Fungal Diversity in the Burrow Wall Soil of Two Earthworms Species

Kavitha B.1, Bagyaraj D.J.

2

1Department of Microbiology, Maharani‘s Science College for Women, India

2Center for National Biological Resources and Community Development, India

Earthworm burrow wall creates a favourable microhabitat for the soil microflora with generally

enhanced microbial activity in the burrow wall soil as compared to bulk soil. Nitrogenous wastes

excreted through the body surface of earthworms accumulate in the burrow wall and affect the soil

microbial community in those areas. Much of the work regarding earthworm effects on

microorganisms has focused on the functional significance of microbial-earthworm interactions.

Virtually nothing is known about the possible effect of burrow wall on the taxonomic composition of

communities of soil microorganism‘s particularly filamentous fungi. The present study focuses on

the comparison of the taxonomic structure of the fungal community and fungal diversity in the

burrow wall soil of two species of earthworms, viz., Lampito mauritii and Pontoscolex corethrurus,

both of which are abundant in most areas in and around Bangalore, India. The soil sample was

generated in the laboratory by maintaining earthworms in chambers with a dimension of 30 cm

height, 40 cm length and 10 cm width. Soil sample were collected by the destructive method at an

interval of 30 and 45 days. The frequency of occurrence of fungal taxa as percentages was calculated

and the community of filamentous microscopic fungi was studied. DMRT showed a significant

increase in the total fungal count in the burrow wall soil P. corethrurus (both 30 and 45 days)

compared to control, where as the reverse was true with L. mauritii. In both the species of

earthworms the 45 day burrow wall soil showed higher total fungal count compared to 30 day soil

sample. A total of 55 fungal species which included 29 genera were isolated during this study. Six

and seven fungal species were isolated only from the burrow wall soil of P. corethrurus and

L.mauritii respectively. Higher species diversity and species evenness was observed in the burrow

wall soil of L. mauritii compared to P. corethrurus.

213


5th to 10


Oral – Session 6

Vermicompost as biocontrol agent in suppression of three soil-borne plant

pathogens at field level

Reddy Shobha A.1, Bagyaraj D.J.

2, Kale Radha D.

1

1 Mount Carmel College, India

2 CNBRCD, Bangalore

Three serious soil-borne plant pathogens vis-à-vis Fusarium oxysporum, Xanthomonas campestris and

Ralstonia solanacearum are causing considerable damage to vegetable crops. These pathogens are difficult to

manage by chemical methods. The current interest is to promote their biological management. The present

study was carried out to evaluate the effect of seed treatment with 10% aqueous extracts of vermicompost

prepared from different substrates [agricultural waste, leaves of Azadiractina indica (neem) Parthenium

hysterophorous (Parthenium) and Lantana camara (Lantana)] and application of vermicomposted neem to soil.

The test crops were tomato (Lycopersicon esculentum) and egg plant (Solanum melongena). Vermicompost

was prepared by mixing the respective substrates with cow dung slurry (9:1 ratio w/w) separately. The seeds of

tomato and egg plant were soaked for one hour in different aqueous extracts of vermicomposts before sowing.

The seedlings were raised in separate nursery beds of size 1m X 1m to which vermicompost was applied at the

rate of 5 tonnes/ha. From the nursery beds seedlings were transplanted to plots and each plant received 250 g of

vermicompost (spot application). The number of plants was 50 per plot. Seed treated with water and Farm Yard

Manure served as control. Plots were chosen where the incidence of pathogens was high (R.solanacearum - 22

X 103 CFU/g; F.oxysporum – 19 X 10

3; X.campestris – 34 X 10

3). The microplots in triplicate for each of the

treatments were monitored for both survival of transplanted seedlings and yield. The results revealed that the

best treatment for suppression of bacterial and fungal wilt diseases in tomato and egg plant is seed treatment (1

hour) with 10% aqueous extract of vermicomposted neem coupled with application of vermicompost to the soil

both during sowing as well as on transplantation. This treatment reduced the incidence of both fusarial and

bacterial wilt by 100%. Better biomass and yield were observed on application of vermicomposted neem. In

tomato crop, the survival was 100% when seeds were soaked in aqueous extract of vermicomposted neem and

in the field receiving vermicomposted neem. The least survival was found in controls where the seeds were

soaked in water before transplanting them in to fields receiving either vermicompost (70 %) or Farm Yard

Manure (58%) respectively. The total yield was recorded for three harvests at time duration of fifteen days.

Among different treatments, the seed treatment with vermicompost extract followed by application of

vermicompost to field resulted in maximum yield (385kg/plot) and least in plot supplied with Farm Yard

Manure (87.46 kg/plot). It was found that the application of vermicompost alone is not enough to protect the

plants against the disease but coupling it with seed treatment is important to achieve complete disease

suppression and to get better yield. Similar treatments were given to egg plant seeds before transplanting the

seedlings to respective plots. The survival was 98% in treatment where seeds were soaked in aqueous extract of

neem based vermicompost that received neem based vermicompost after transplantation. The least survival of

22 % was encountered in the control treatment with seeds soaked in water and transplanted to field receiving

Farm Yard Manure. The total yield was recorded for two harvests at time duration of 20 days. The lowest yield

was in plot supplied with Farm Yard Manure.

214


5th to 10


Oral – Session 6

Growth and reproduction in Pontoscolex corethrurus (Muller) – a

dominant species of rubber plantation

Nath Sabyasachi, Chaudhuri Priyasankar S.

Department of Zoology, Maharaja Bir Bikram College, India.

Pontoscolex corethrurus, an exotic geophagous earthworm with wide range of tolerance to environmental factors, is

the most dominant earthworm species in the soils of rubber plantations in Tripura (India). Under laboratory condition, it showed highest growth and reproduction rates compared to native geophagous species of earthworms

when subjected to same diet (unpublished data). So laboratory experiments were set up in order to evaluate the effects of different food substrates (collected in and around the rubber plantation sites) on the growth and

reproduction of this tropical earthworm. Decomposed five different experimental diets used were cowdung, rubber

leaf litter (Hevea brasiliensis), rubber saw dusts, bamboo leaf litter and mixture of these plant residues. All the experimental diets included mineral soil as the main substrate. The chosen materials were mixed with soil in different

proportions to provide five treatments (dry weight proportions in brackets): 1) Soil-cowdung (30:2); 2) Soil-cowdung-rubber leaf litter (30:1:1); 3) Soil-cowdung-rubber saw dusts (30:1:1); 4) Soil-cowdung-bamboo leaf litter

(30:1:1) and 5) Soil-cowdung-mixed diet of rubber leaf litter, rubber saw dusts and bamboo leaf litter (30:1:1). Soil-

cowdung mixture was considered as control and the rest as experimental diets. Cowdung was introduced in all the treatments to increase the suitability of the culture media, as suggested by Lowe and Butt (2005). In each treatment

(three replicates) one pair of young individuals (P. corethrurus) of similar weight and age group was inoculated

randomly per plastic pot (15×15×8 cm) containing 1600 g substrate. The culture pots were kept in BOD at a temperature of 27ºC and 25% moisture of the substrate was maintained. The experiment continued for 105 days and

the culture media kept unchanged till the end of the experiment. Biomass (fresh weight) and juvenile number were recorded every 15 days. In the first fortnight, biomass value increased to more than double with the appearance of

clitellum in all the experimental diets. Maximum weight for the endogeic P. corethrurus was observed in both soil-

cowdung-bamboo leaf litter and the diet mixture having rubber leaf litter, rubber saw dusts and bamboo leaf litter, whereas minimum weight was obtained in the soil-cowdung treatment (control). Highest growth rate was achieved in

the mixture with rubber saw dust (21.14 mg worm-1

day-1

) which however was not significantly different (p>0.05)

from that in the mixture having rubber leaf litter, rubber saw dusts and bamboo leaf litter (20.92 mg worm-1

day-1

), bamboo leaf litter (20.27 mg worm

-1 day

-1) and rubber leaf litter (18.81 mg worm

-1 day

-1). Lowest growth rate was

noticed in the soil-cowdung mixture (13.36 mg worm-1

day-1

) which was significantly lower (p<0.05) than that in all other treatments. Lowest and highest fecundity rates were obtained in the soil-cowdung (0.35 juveniles adult

-1 week

-

1) and in the mixture with rubber saw dust (1.21 juveniles adult

-1 week

-1) respectively. Fecundity rate in the latter was

however not significantly different (p>0.05) from that in the mixture with bamboo leaf litter (0.94 juveniles adult-1

week

-1) and diet mixture of rubber leaf litter, rubber saw dust and bamboo leaf litter (0.89 juveniles adult

-1 week

-1).

Fall of biomass of P. corethrurus in most of the diets after 90 days was due to reproductive costs as reflected by dramatic increase in juvenile production in all the diets after 105 days. In fine, P. corethrurus showed higher rate of

growth and reproduction in the treatments with saw dust or bamboo leaf litter or in a combination of both. Because of

least growth and reproduction rate in cowdung, it is not considered to be a food of choice for its culture. Thus our results indicate that for vermiculture of P. corethrurus to be used as a source of protein or for its introduction and

systematic use in low-input tropical agriculture (as suggested by Lavelle et al.1987), it is necessary to add suitable

bulking agents like saw dusts, bamboo leaf litter etc. in the soil in order to maintain its successful field population.

215


5th to 10



Soil enzymatic activity on presence of Pontoscolex corethrurus

(Oligochaeta: Glossoscolecidae), Cyclocephala barrerai and Phyllophaga

setifera (Coleoptera: Melolonthidae)

Alarcón Gutiérrez Enrique1, Romero López Angel Alonso

2

1 Instituto de Investigaciones Forestales Universidad Veracruzana

2 Instituto de Ecología, A.C

Soil enzyme activity in the presence of adult earthworms of Pontoscolex corethrurus Muller and late

instars of Cyclocephala barrerai Martínez and Phyllophaga setifera Burmeister (―white grubs‖) was

evaluated. The substrate was an agricultural soil mixed with 1.5% of dried mucuna (Mucuna

pruriensis var. utilis, Fabaceae) as an organic matter. The experimental test was completely

randomized, with four treatments and six replications of each: treatment I, control, without

individuals; treatment II, with three individuals of C. barrerai; treatment III, with three individuals of

Ph. setifera and treatment IV, with three adult earthworms of P. corethrurus. All the containers

stayed to at 24 ± 2°C, constant illumination (25 watts) and relative humidity of 65 ±5%. Enzymatic

activity was evaluated from each treatment, at initial (t0) and final (day 30th

; t30) time. Soil samples

(50 g) were taken from 10 cm depth of each container, placed under plastic bags and stored at 4°C

until analysis. The activity of fluorescein diacetate hiydrolase (FDA, total esterase test) was

determined according to Alarcón-Gutiérrez et al. (2008). No significant difference (Mann-Whitney

test, p> 0.05) was observed between treatments when different times were evaluated; although there

was a tendency to major activity in treatment II (0.0041nkat/g DM) with respect to the other

treatments. Nevertheless, when sampling time was compared (t0 versus t30), in the same treatment,

significant difference (Mann-Whitney test, P<0.05) was detected. Results indicate that soil enzymatic

activity in the presence of Pontoscolex corethrurus (0,0006 ± 9.3 x 10^-5 nkat/g DM ) is similar to

that observed in the soil of white grub species: Cyclocephala barrerai (0,00049 ± 1.5 x 10^-4 nkat/g

DM) and Phyllophaga setifera (0,00059 ± 1.8 x 10^-4 nkat/g DM) after 30 days of experiment.

216


5th to 10



Evaluation of the effect of fodder trees and earthworms on the

growth of sorghum

Bautista Francisco1, Ávila Chi Marco Antonio

2, Meléndez Ramírez Virginia

2,

Huerta Esperanza3

1 UNAM

2 Universidad Autónoma de Yucatán

3 El Colegio de la Frontera Sur, Unidad Villahermosa

In Yucatan, Mexico, the land that initially had tropical deciduous forest is now grassland and now about

1.5 million hectares are disturbed. The extensive livestock farming by slash and burn are of low productivity due to shallow soils and climate of humid tropics and high degree of disruption of

ecosystems. Sylvopastoral systems (SSP) are an agroforestry management option for livestock purposes its settlement is affordable and promotes conservation and several environmental services which can coexist with traditional livestock, although to promote greater technical knowledge is required. There are

about 211 forage species used by the Mayans, which can be used for the design of SSP prior recognition. In addition, are well known the benefits of earthworms in soil fertility modifying many soil properties, which affect plant growth and therefore its production. This work was done in search of design options for

agroforestry systems where forage species benefit to plants and the macroinvertebrate community for evaluating the effect of the quality of litter species of fodder trees combination with the earthworm on

growth of sorghum (Sorghum vulgare) as an indicator of soil fertility. Thus, under conditions of greenhouse, it was analyzed the effect of the quality of the foliage of six species of trees, G. floribundum (G), B. simaruba (B), P. piscipula (P), D. cuneata (D), C. gaumeri (C) and R. longiloba (R) and the

earthworm Dichogaster bolaui in the growth of the sorghum and the earthworms populations. Plastic pots were used containing 2 kg of soil, mixed with 100 g of dry foliage of each used tree and 1.2 g of earthworms biomass was applied to the pot. The design was completely randomized and the information

was analyzed by analysis of variance and principal components analysis to know the relation among the quality of the foliage and the growth of the sorghum and the biomass of earthworms. The foliage of B and

G that contain the highest amounts of phosphorus allowed a better production of dry matter of the sorghum, though the better height of this plant was correlated with the highest amounts of neutral detergent fiber. The foliages of B and G with or without earthworms benefit the growth of the sorghum.

The application of earthworms produced an increase of the total biomass and the height plant of sorghum. The foliages of R and C favour the populations of D. bolaui but not the growth of the sorghum. In the group of treatments with earthworms, the foliage of P. piscipula and D. cuneata benefited earthworms

populations, but they did not benefit the growth of the sorghum. This study also found that there are tree species such as R and C that can benefit the soil fauna, as the worms, immediately by releasing nutrients more easily helping to initiate the cycle of organic matter. Thus, when designing agroforestry systems is

the combination of tree species to encourage diversity in the quality of the substrates for systems to be environmentally stable.

217


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Estimating automatically the abundance of belowground earthworm casts:

dream or reality?

Bottinelli Nicolas1, 2

, Hallaire Vincent1,2

, El Housni H.1, 2

, Menasseri-Aubry S.1, 2, 3

,

Cluzeau Daniel4

1INRA, France

2Agrocampus Ouest, France

3Université européenne de Bretagne, France

4Université Rennes 1, France

Belowground earthworm casts have a profound influence on soil processes. To predict the effects of

earthworms on soil quality, ecologists need adequate methodologies to assess earthworm casts. We

attempted to estimate belowground earthworm casts by image analysis. First, we compiled a learning

set of three types of pores organized between: (i) individual casts (PIC), (ii) coalescent casts (PCC)

and (iii) unknown aggregates (control pores). Then, each type of pore was classified according to two

shape indexes. Second, we applied this model on soil blocks sampled two weeks after sowing maize

under moldboard plowing (MP) and no-tillage (NT), at 2- and 12-cm depths. Third, we compared

results from our new model with results obtained by point counting on individual casts. According to

the model, The PCC were well discriminated from the other pore types, while the PIC were

confounded with the control pores. Hence, our model was thereafter only used to assess the PCC

abundance under the two tillage treatments. Results indicated that just after tillage almost all PCC

were destroyed under MP; as consequences the PCC abundance was much higher under NT

compared to MP for the both depths. The PCC abundance was positively correlated with the

abundance of individual casts (r= 0.68, p<0.01) demonstrating that our model is relevant to

automatically estimate the abundance of belowground earthworm casts. This automatic method may

allow for more effective evaluation of earthworms influence on soil quality.

218


5th to 10



Life history studies of Lumbricus friendi (Cognetti, 1904)

Butt Kevin R.1, Briones Maria J.I.

2


2 University of Vigo

The objective of this work was to obtain baseline data on the life cycle of the anecic earthworm

Lumbricus friendi, which although known to science for over a century is little studied. Field-

collected specimens from NW Spain (where this species is widespread) were grown to maturity and

maintained under controlled laboratory conditions. Thereafter experiments were undertaken to

determine its mode of reproduction, reproductive output, cocoon development and juvenile growth

rate. Effects of density and temperature on earthworm growth were also examined. Plastic culture

vessels containing a substrate of Kettering loam were incubated in temperature-controlled chambers,

with dried horse manure as food. Paired, mature animals were sampled monthly over a period of 9

months for cocoon production. Cocoons were incubated on moist filter papers in Petri dishes and

hatchability and hatchling growth rates were recorded. Results showed that at 15 ºC individuals

matured at a live mass of 3.5 g. Zero cocoons were produced by virgin animals maintained in

isolation but recently-matured, paired L. friendi produced 2.16 cocoons worm-1

wk-1

. Cocoons were

lemon-shaped with a smooth pale yellow-coloured outer surface and a mass ranging between 17 and

37 mg. At 15 oC, cocoons required around 3 months to hatch and hatchability from the first five

months of cocoon production was 85 % successful. The majority of cocoons produced a single

hatchling although twins were also occasionally recorded. Growth to maturity of these hatchlings was

achieved within a period of 5 months, with tubercular pubertatis recorded after 3 months. Increased

temperatures in the range of 5 – 15 oC led to significantly increased growth rates, whereas increased

earthworm density led to a significant decrease in rate of growth. These findings permit comparisons

with closely related, but more closely studied species such as Lumbricus terrestris.

219


5th to 10



Earthworms from an alkali industry waste site in North West England

Butt Kevin R.

1, Briones Maria J.I.

2


2 University of Vigo

Soils at this post-industrial site have developed over the past century from waste alkaline materials

(pH 12) dumped to a depth of some 10 m. These soils, now suitable for plant growth, were further

contaminated historically by acidic boiler waste in some areas and retain a pH of 4. The site, near

Bolton, is designated a Site of Special Scientific Interest (SSSI) for the well documented flora,

dominated in spring by orchids. However, practically nothing is recorded of the site‘s soil fauna. The

objectives of this research were to determine which earthworm species were present, to measure

densities and biomasses and how the community composition varies horizontally along the pH

gradient. Finally we aimed to determine potential earthworm origin of the colonising animals. The

shape of the 9 ha site, surrounded on 3 sides by the confluence of 2 rivers and a disused canal, meant

that this could be assessed by sampling in linked, adjacent habitats of woodland, grassland and

further ex-industrial areas. Preliminary investigations were undertaken in autumn 2008 to assess

earthworm community composition by sampling (hand-sorting of soil plus mustard vermifuge

application) at areas differing in soil pH and site management: (i) acidic, (ii) alkaline unmanaged and

(iii) alkaline managed by scrub-cutting to retain the desired flora of the SSSI. The following autumn,

a 110 m transect was sampled between extremes of soil pH. Initial results revealed a total of 7

earthworm species including the two anecics, Aporrectodea longa and Lumbricus terrestris, of

interest as the depth of habitable soil, above the weathered alkaline material is less than 20 cm. Scrub

management had a positive effect on earthworm density (204 m-2

) compared with unmanaged areas

(134 m-2

) in the alkaline soils. Acidic soils (pH 4.7) supported a lower diversity of earthworms,

consisting mainly of L. rubellus and L. castaneus - average density 64 m-2

. Four further earthworm

species were revealed in subsequent work, including Dendrodrilus rubidus in the acidic areas. A.

caliginosa accounted for more than half of the earthworms located across the site and was found in

all but the most acidic soils. Sampling in soils surrounding the site showed all but two of the (least

common) species found on site, thereby acting as a potential source. Results show that earthworms

are able to colonize and survive in the shallow soils above the inert material of what was once an

uninhabitable environment.

220


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Reproductive Strategies of Tropical Earthworms from Tripura (India)

Chaudhuri Priyasankar S., Bhattacharjee Subhalaxmi, Bhattacharjee Gautam

Department of Zoology, Maharaja Bir Bikram College, India

Some data on the reproductive biology of thirteen tropical earthworm species under fluctuating

laboratory conditions are presented. The culture pots (4.5 L) received one pair of clitellate earthworm

species and varying amounts of air-dried ground and sieved (0.05 mm mesh) field soil mixed with

15-day old cow dung dusts (particle size < 2mm) as food substrate except for Perionyx excavatus,

where only air dried cow dung dusts were used. The moisture level of the substrate was maintained

around 70% and 40% for P. excavatus and other species respectively by periodical spraying of water.

Cocoons were collected by gentle wet sieving through 0.05 mesh sieve. The number of cocoons

produced was calculated on a per individual basis. After isolation, cocoons were kept on moist filter

paper spread over water soaked cotton (85% moisture) inside a petri-dish (one cocoon per dish) to

study the incubation period and hatching pattern of juvenile worms. The newly hatched juveniles (F1)

of the studied earthworm species were kept individually in fresh culture and reared until they became

clitellate and started to produce cocoons. Copulation is not prerequisite for production of viable

cocoons in Pontoscolex corethrurus, Dichogaster affinis, Metaphire houlleti, Octochaetona beatrix

and Polypheretima elongata, indicating that these worms may be parthenogenetic. Continuous

breeding with high fecundity (cocoon production worm-1

year-1

) was recorded in peregrine

earthworms viz. P. excavatus (156 cocoons worm-1

year-1

), P. corethrurus (118), Dichogaster

modiglianii (68), D. affinis (46), Lampito mauritii (43) and O. beatrix (40). Native earthworms are

either semi-continuous breeder with moderate fecundity (Drawida nepalensis 29, Drawida

assamensis 11, Drawida papillifer papillifer 11) or discrete breeder with least fecundity (Eutyphoeus

gammiei 1, Eutyphoeus comillahnus 2). Incubation period (days) was low in P. excavatus (13), D.

modiglianii (14), L. mauritii (15), D. affinis (21), D. papillifer papillifer (21), D. nepalensis (24), D.

assamensis (29), P. corethrurus (29); moderate in P. elongata (49), O. beatrix (49) and high in E.

comillahnus (51), M. houlleti (64) and E. gammiei (110). Hatching success (%) was high in P.

corethrurus (91), M. houlleti (91), D. affinis (81), O. beatrix (81), D. modiglianii (63), L. mauritii

(60), P. excavatus (52) and moderate to low in P. elongata (40), D. nepalensis (37), D. assamensis

(33), E. gammiei (20) and D. papillifer papillifer (18). More than one hatchling per cocoon was

recorded in L. mauritii, D. nepalensis, P. elongata, D. assamensis and M. houlleti. There was a

dramatic increase in cocoon production by most earthworm species during summer and monsoon in

contrast to the winter. With rise in temperature, incubation period increased in P.corethrurus, P.

elongata, D. nepalensis, E. comillahnus and O. beatrix and decreased in P. excavatus, M. houlleti, D.

affinis, D. modiglianii and D. assamensis. High rate of cocoon production, short incubation period

with higher hatching rate as well as continuous breeding strategies in P. excavatus, P. corethrurus, L.

mauritii, D. affinis, D. modiglianii, O. beatrix indicate their possible usefulness in vermiculture either

for waste degradation or for soil reclamation depending upon the species.

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Conventional tillage and sampling date effects on earthworm population in

a maize monoculture in Central Mexico

De León González Fernando1, Rosas Miguel Angel

1, Flores Macías Medina Antonio

1, Payán

Zelaya Fidel1, Fragoso Carlos

3, Ramos Espinosa Guadalupe

1, Nava Rodríguez Verónica

1,

Gutiérrez Rodríguez Francisco2, Borderas Tordesillas Fernando

3

1 Universidad Autónoma Metropolitana-Xochimilco.

2 Universidad Autónoma del Estado de Mexico.

3 Instituto de Ecología, A.C.

An earthworm population of Aporrectodea caliginosa was analysed on three sampling dates (maize

emergence in spring, crop harvest in autumn and post-harvest in winter) under four tillage systems:

1) shallow tillage; 2) normal ploughing; 3) ripper decompaction (one pass); and 4) ripper

decompaction (two passes). We also considered two soil depths (0-15, 16-30 cm). The soil was a clay

loam in under irrigation in Central Mexico. The tillage systems were applied during four consecutive

years on experimental plots (400 m2 area) cultivated with maize. The tillage treatments did not

provoke differences in juvenile, mature and total earthworm individuals. In contrast, the sampling

date had a significant effect (P<0.05) on the earthworm population: individuals were < 10 m-2

at

maize emergence (in April); 135 m-2

at harvest (in November); and 45 m-2

at post-harvest (in

January). Sampling date also affected the earthworm biomass. Earthworm abundance showed

differences caused by soil depth, but only on the post-harvest date, when individuals apparently

migrate to the deep layer; individual populations were higher in the 16-30 cm soil layer than in the 0-

15 cm soil. Burrowing activity was not affected by tillage treatments or by the sampling date.

Burrows were constant in three sampling dates, suggesting that the bio-structures created by

earthworms resist the erosive impacts of irrigation, rainfall, and tillage implements. Soil moisture and

soil resistance changed in the three sampling dates, while the soil organic matter fractions remained

constant. Juvenile and adult individuals on the maize emergence date were significantly associated

(P<0.05) with heavy macro-organic matter. The lack of negative effects of intense tillage sequences

on the earthworm population suggests that other factors acting together (2-5 ton ha-1 year-1 of maize

stover restitution, clayey texture, high soil water content due to irrigation) determine the dynamics of

earthworm population.

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Are vermicompost and the stimulation of endogeic earthworm activities

relevant alternatives to chemical fertilizers?

Doan Thu Thuy

Soils and Fertilizers Research Institute (SFRI- Hanoi)

Soil degradation and water pollution are widespread land degradation problems in Vietnam. There is

therefore an urgent need for sustainable agricultural practices. Presently, farmers and researchers are

investigating different strategies to maintain soil fertility and avoid off-site effects. Within this

context, direct (i.e., the drilosphere) and indirect (vermicompost) earthworm activities could be

considered as interesting ecosystem services suppliers. The aim of the present study was to examine

the interactions between an endogeic earthworm species, Metaphire khami, compost, and

vermicompost produced by Eisenia andrei, an epigeic earthworm species, in a degraded tropical soil.

A year-long experiment was carried out in greenhouse with a corn-tomato-corn cycle. Following

organic fertilizer inputs earthworms survived and soil properties were improved; the soil‘s carbon

and nutrient contents were higher. For the first corn crop, the same yields were obtained with

vermicompost inputs as with chemical fertilizer. However, this positive effect did not continue with

time and no difference was observed between vermicompost and compost for the second corn crop,

one year later. The effect of M. khami on corn growth was unexpected. While we observed a positive

interaction between M. khami and vermicompost, the earthworms had a negative impact on plant

growth in combination with compost. In conclusion, this experiment demonstrated that organic

matter amendments do show interesting potential for the rehabilitation of tropical soils. However,

negative interactions can occur between local endogeic earthworms and compost.

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Changes in soil quality after passage through the gut of Pontoscolex

corethrurus (Oligochaeta: Annelida)

Duarte Ana Paula, Vander de Freitas Melo, Pauletti Volnei, Brown George

Universidade Federal do Paraná Brazil

Earthworms and a few other groups of large invertebrates have the ability to ingest soil altering its

physical and chemical characteristics. To evaluate the effect of earthworms on soil properties in a

regenerating mine-site, three soil types (Neossolo Litólico - RL, Cambissolo Háplico – CX and

Neossolo Quartzarênico - RQ), differing in clay and organic matter contents were collected from a

lead-mining area in Adrianópolis-PR, Brazil, at two depths: 0-10 and 20-40 cm. Earthworms

(Pontoscolex corethrurus) were collected from the Federal University of Paraná Farm and five adults

were incubated per container (n=3 replicates) in the three soils (300 cm3), maintained at 70% field

capacity and at room temperature. Castings and control soil were collected every two days using

tweezers and the analyses were performed on uningested soil (control) and the casts. Parameters

analyzed were: pH-H2O, exch. acidity, CEC, exch. Ca, Mg and Al, available K and P, total organic

carbon (TOC) and nitrogen (TN), soil texture and water dispersible clay. The results were submitted

to ANOVA using SAS and the means were compared by Tukey test (p <0.05). The worms selected

soil fractions to ingest, increasing fine sand and silt, reducing total sand contents. They also altered

soil fertility, increasing levels of available P and K, CEC and Ca+Mg. Few effects on TN were

observed, TOC increased only in the castings from the poorer soil (selection of C-rich particles) and

pH was generally higher in the casts. The only side effect observed due to gut passage, was the

increase of clay dispersed in water, which can increase soil erodibility.

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Laboratory and field studies on the lifecycle of Dendrobaena alpina

(Rosa, 1884)

Kostecka Joanna1, Butt Kevin R.

2

1 University of Rzeszow,


Distribution of Dendrobaena alpina covers the mountainous region of central Europe. It is

considered an epigeic or intermediate earthworm with a high dominance and frequency index. In the

field it is easily recognized among other species because of its violent escape response. This work

describes data from field studies conducted over an annual cycle in the Bieszczady National Park in

different types of beech (Fagus) forest. Additionally we set out to obtain baseline data on the life

history of D. alpina under controlled environmental conditions. Animals were collected from forest

soils near Lutowiska in the Carpathian Mountains, SW of Poland in June 2008 and taken to the

laboratory for investigation. All were initially housed individually in native soil within plastic

containers, but over time soil was replaced with a standard Kettering loam. Food was supplied in the

form of dried horse manure. Mature individuals were kept in isolation and cocoon production

monitored on a 2 monthly basis. Immature individuals were kept until they reached maturity, at

which point they were put into pairs and again combined output of cocoons regularly monitored.

Cocoons themselves were collected, had masses determined and were incubated on moistened filter

papers in Petri dishes. Hatchability was recorded, as was estimated duration of incubation and growth

to maturity. Field-collected adults had a mean mass of 1.40 g. Results over 21 months at 15 ºC,

revealed that these individuals (n=6) produced an average of 1.83 cocoons worm-1 month-1. Zero

cocoons were produced by further animals, collected as immatures and grown to maturity and

maintained in isolation. However, recently-matured (mean mass 1.32 g), paired D. alpina produced

an average of 1.65 cocoons worm-1

week-1

(9 pairs over 15 months). All individuals and pairs were

kept in 0.4 litre pots. Cocoons had a mean mass of 19 mg (range 14 - 26 mg). At 15 ºC, cocoons

produced by recently matured adults during the first 2 months, hatched after approximately 80 days

(hatchability of 82 %). Thereafter the majority of cocoons, although showing visible development of

a viable earthworm, failed to hatch which may be an anthropogenic function. Hatchling growth from

an average of 18 mg to maturity (at 1.2 g) required 7-11 months in the given substrate with

Tubercula pubertatis seen after 5 months. This work presents both field and laboratory-based data on

this particular species.

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Earthworm abundances under different agricultural managements in a

kiwi fruit plantation

Lago M.C.F., Gallego P.P., Briones Maria J.I.

Universidad de Vigo, Spain

Sustainable agriculture improves soil fertility and health. Consequently, it is becoming a feasible

alternative to current intensive cropping practices. However, in northern Spain kiwi fruit (Actinidia

deliciosa) is still mostly produced in intensively managed plantations. Earthworms play an important

role in soil fertility; however, agricultural practices have detrimental effects on their populations and

higher mortality rates have been often recorded. In this study, we investigated the effects of four

different management practices, typically used for producing kiwi fruits in Galicia (namely

conventional (CT), ecological (ECO) and two integrated systems (INT- and INT+)), on earthworm‘s

diversity and abundance and soil properties (soil pH, organic matter and soil moisture contents).

Sampling was performed at three different growth stages of the kiwi plant: (i) resting period, (ii)

sprouting and (iii) fruit growth. On each sampling occasion, 15 intact soil cores (11 cm diam.x16 cm

deep) were taken from each of these management treatments. The cores were sliced horizontally into

2 cm layers down to a total depth of 10 cm; sub-samples from each layer were used for chemical

analyses (%C and %N) and soil moisture determinations. In addition, three soil samples

(50x50x10cm) were also randomly taken for earthworm and pH determinations. In total, eight

earthworm species were identified: Lumbricus friendi (average density of 63m-2

), Octolasion lacteum

(25m-2

), Aporrectodea caliginosa (19m-2

), Dendrobaena octaedra (6m-2

), D. madeirensis (4m-2

),

Amynthas sp. (4m-2

), L. rubellus (3m-2

) and Eisenia fetida (1m-2

). Results also showed that INT- and

ECO treatments supported the highest species number and abundances. In addition, abundances of L.

friendi and O. lacteum showed a positive relationship with soil pH (R=0.55 and 0.48, respectively),

whereas D. octaedra preferred lower values (R=0.39). Furthermore, L. rubellus populations were

negatively affected by nitrogen content. Finally, soil moisture did not play a significant role possibly

be due to the fact of all treatments having automatic irrigation systems. In conclusion, our results

confirm that agricultural managements have important impacts on earthworm populations. In

particular, at the sprouting period when the plots (excepting the ecological) received herbicides and

consequently, organic matter dynamics and soil pH were altered, earthworm‘s abundances and

diversity were mostly affected.

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Production of tomato saladette in greenhouse: a combined effect of

earthworms and green manure

López-Hernández Dante J.1, Cid-Santiago Carlos A.

1 Noa-Carrazana Juan C.

2, Ortiz-

Ceballos Gustavo C.3, Ortiz Ceballos Angel I.

2

1 Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana. Mexico

2 Instituto Tecnológico de la Cuenca del Papaloapán. Mexico

3 Facultad de Ciencia Agrícolas, Universidad Veracruzana. Mexico

It has been documented the activity of earthworms and green manure promoted the growth and

increased the annual and perennial cultures production. Nevertheless, the knowledge in protected

culture is limited. In these intensive production systems pesticides are applied in the ground and on the

plants. However that can be toxic for the survival and development of earthworms. Also, it is known

that the nitrogen fertilizers can be replaced by green manure to incorporate to the soil organic matter

rich in nitrogen, and to reduce the production costs. Because of its ample distribution and tolerance to

disturbed atmospheres, the earthworm Pontoscolex corethrurus has been the tropical species more

studied and handled to foment the growth and agricultural production. The present study had as

objective to study the effect of the presence/absence of the exotic earthworm P. corethrurus and the

leguminous named picapica (Mucuna pruriens var. utilis) in production of tomato saladette. In

greenhouse, during a period of 230 days, 80 plants of tomato, 20 plants were cultivated by treatment.

The evaluated treatments were: a) conventional handling without earthworms and mucuna (-E-M);

with earthworms and without mucuna (+E-M); without earthworms and with mucuna (-E+M) and with

earthworms and mucuna (+E+M). At the end of the experiment the earthworm and its interaction with

the green manure (mucuna) affected the number significantly (F = 18.49, P < 0.0001; F = 4.16, P <

0.04) and biomass (F = 27.86, P < 0.0001; F = 11.67, P < 0.001) of the fruits. The greater production

of tomato (number and biomass) was registered in the treatments with absence of earthworms (-E-M)

and (–E+M). Nevertheless, when they incorporate together (+E+M) to the soil an intermediate

production was obtained. We conclude that the addition of green manure and earthworms can be an

alternative for an organic and low costs production..

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Impact of management of the production systems of maize in the

populations

Ramírez Gómez Salvador1, Córdova Avalos Victor

1, Palma-López David J.

1

Ortiz Ceballos Angel I.2

1 Colegio de Postgraduados- Campus Tabasco. Mexico

2 Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana. Mexico

The key factors for the earthworms indirect management is the continual supply of organic matter,

reduced tillage, and avoiding the use of pesticides and fertilizers are toxic to earthworms. This study

was performed in the southern of Mexico in the summer cycle and aimed to assess the impact of the

management of four maize production systems (Fallow, Mechanical tillage, Transition and Organic)

in populations of earthworms. It registered the diversity, abundance and biomass of earthworms in

each production system of maize. Each system had three replicates based on a randomized design.

The results showed that the earthworm populations consisted of two native species (Larsonidrilus

orbiculatus and Lavellodrilus sp.) and an exotic (Pontoscolex sp.). In all system, the dominant

species were natives. The total biomass was significantly higher in Transition Systems and Organic

maize than Fallow and Mechanical tillage. We conclude that mechanical tillage had a negative

influence in the populations of earthworms.

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Influence of the earthworm Pontosocolex corethrurus in the survival and

growth of Abies religiosa in the nursery stage

Sánchez Velásquez Lazaro Rafael, Vásquez-Morales Suria Gisela, Lara-González Rogelio,

Ortiz Ceballos Angel I.

Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana. Mexico

In the forest nursery of Mexico the plastic bags are still widely used for the massive production of

seedlings. It is suggested that the constant irrigation alters the soil conditions (for example, the

compaction) that affect the growth and development of the roots. It is known that the earthworms can

improve the quality of the soil (physical, chemical and biological) and are of easy management.

Nevertheless, there are no studies on the effect of earthworms on the survival and growth of

seedlings Abies religiosa. In the present research the effect of earthworms Pontoscolex corethrurus

was evaluated in the survival and growth of seedlings Abies religiosa. In greenhouse conditions,

during a period of 365 days, seedlings were cultivated in plastic cubs under three treatments (0, 1 and

2 juveniles earthworms), with 25 replicates by treatment. Every 15 days registered the height,

diameter of the stem, maximum and minimum cover and number of branches. In addition, when

finishing the experiment it was determined the total dry matter production and by component of

plants (root, stem, branches and leaves). The results showed that the presence of P. corethrurus had a

significant effect when increasing height of the plant and dry matter production (total, root and stem).

It is concluded that the presence of earthworms promotes the growth of seedlings.

229


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Temporary effect of burning on earthworm communities and physico-

chemical properties of soil in areas of Eucalyptus grandis

Uribe López Sheila Ariany1, 2

, Huerta Esperanza1, Geissen Violette

1, Godoy Roberto

3,

Dendooven Luc4, Jarquin Aaron

1

1Departamento de Agroecología, El Colegio de Frontera Sur, unidad Villahermosa. Carr. Villahermosa-

Reforma km. 15.5. Ra. Guineo 2da. Secc. C.P. 86280. Villahermosa, Tabasco, Mexico. 2UNIVERSIDAD POLITECNICA DEL CENTRO. Río Usumacinta No. 112, col. Casa Blanca 1era. Secc. C.P.

86060. Villahermosa, Tabasco, Mexico. 3Instituto de Botánica, Facultad de Ciencias. Universidad Austral de Chile, Casilla 567- Valdivia.

4CINVESTAV- MEXICO.

The town of Huimanguillo, Tabasco, counts on an extensive zone of eucalyptus (Eucalyptus grandis)

plantations, because it generates significant revenue sources for the small producer, to lease their land

to transnational corporations in the area of wood and cellulose. However, several studies conducted

in other tropical areas suggest that by introducing this crop acidify this soil, compete with native

flora, produce allelopathic effects, absorbs large quantities of water to the soil, etc. This study aims to

evaluate the temporal effect of burning in management of Eucalyptus grandis sites on the

communities of earthworms and soil properties, because the presence of these worms indicates the

fertility of soils. The collection of earthworms was carried out at the end of the rainy season in the

month August 2007. Sites were selected to evaluate four treatments: T1 = E. grandis with five years

of age (Euc), T2 and T3 = sites burned 15 to 60 days post-harvest (Q15 and Q60 respectively) and T4

= acahual with more 15 years of abandonment (Acah). The sites had an area of 1-2 ha, in each one

made three quadrants of 50 x 20 m2, and each quadrants six monoliths of 25x25x30cm (TSBF

method). The soils of the area are Acrisols, showed a pH of 4.5-6.0 in the first 30 cm deep. The

nutrients in the soil decreased in the recently burned sites (organic matter, 6-8%, N, 0.19-0.22%) but

once you set the vegetation tends to increase their levels (MO, 9-11%; the N, 0.27-0.33%). Only the

specie found Pontoscolex corethrurus throughout the area, their populations were found in juvenile

stage with highest densities and biomass in the culture of Euc (164.4 ind.m-2

and 36.8 g.m-2

respectively), followed by Acah (138.7 ind.m-2

and 19.1 g.m-2

respectively), while the Q15 and Q60

sites had small populations (80% and 77% with respect to Euc and Acah). In the post-harvest, the use

of burning is part of the techniques used to eliminate the vegetal remainders more easily, however, its

use has negative effects when increasing the risk of losing the ―pool‖ of soil nutrients for the

following cultivation cycle, to compensate this decrement must apply external inputs (chemical

fertilizers) periodically to maintain the crop production, in addition, the management from the

ecologically significant impact on the oligoquetofauna native for the loss of species diversity, soil

fertility decreases and moves and controls the native flora. The state forest industry should

considered to maintain prolonged periods of rest in these areas, which would retain nutrients in the

soil that would result in greater profitability in the medium and long term.

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Food and habitat preference of Lumbricus terrestris L. for cover crops

indicate short-term soil biofumigation effects of the cruciferous Yellow

mustard (Sinapis alba L.)

Valckx Jan1, Cordón Pina Andrea

2, Govers Gerard

1, Hermy Martin

1, Muys Bart

1

1 Katholieke Universiteit Leuven Belgium,

2 Universidad Salamanca, España

Worldwide, cover crops are used because of their multiple roles in soil erosion control, nitrogen loss

reduction, weed suppression, and soil fertility in agro-ecosystems. Recently, (cover crop) species of

the Brassicaceae family have been reported to act as biofumigants against a wide variety of soil-

borne pests (e.g. fungi, nematodes, and slugs) through the release of isothiocyanates (ITC‘s) from

glucosinolate precursors upon cell tissue disruption (e.g. after soil cultivation, freezing). Although

these allelochemicals have the potential to reduce the need for application of synthetic pesticides,

their effects on beneficial soil ecosystem engineers such as earthworms (Lumbricidae) have not been

assessed yet. The common use of mustard powder (variably composed of grinded seeds of Brassica

juncea, Brassica nigra, Sinapis alba) in earthworm sampling may suggest adverse effects on

earthworms. Food preference for five commonly used cover crops (ryegrass (Lolium multiflorum

LAM.), phacelia (Phacelia tanacetifolia BENTH.), oats (Avena sativa L.), and the cruciferous

Yellow mustard (Sinapis alba L.) and rapeseed (Brassica napus L.)) were studied both in food choice

chambers in the lab for 10 days and by litter bags (mesh sizes 0.5 and 4 mm) in a no-till field during

12 months. The effects of growing plants and freshly incorporated residues of three-months-old

Yellow mustard and oats on the habitat preference of adult L. terrestris were studied in simple habitat

preference units consisting of flowering pots connected through a central corridor area in which

earthworms were introduced. Preferences were analyzed by compositional analysis. In general,

ryegrass residues were a preferred food resource over mustard, phacelia or rapeseed residues, and

these were in turn more fed on than oats. Litter disappearance from the 4 mm bags indicated the same

trends for the first two weeks, but then food preference in the field became variable. Between 24 and

48h after incorporation of mustard residues, release of isothiocyanates affected habitat selection by L.

terrestris, but 72h after incorporation these negative effects were neutralized again by ITC

breakdown and volatilization. Habitat selection was not affected by growing mustard plants during

the 12-day study period. The experimental results indicate that Brassicaceae residues are used as a

food resource by earthworms in amounts comparable to other cover crops. Further, neither surface

applied residues nor the living root system of the commonly used cover crop Yellow mustard have

lasting negative impacts on earthworm habitat selection. However, it is strongly advised that mustard

residues are only superficially worked and not ploughed into the soil to stimulate volatilization of

detrimental isothiocyanates.

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Improvement of vermicompost on main nutrients and microbial

population in tomato soil under continuous cropping system: experiment

in laboratory

Zhang Junying1,2

, Xu Yongli1,2

, Li Fu-ping1,2

, Han Dong-yun1,2

, Huang Kai1,2

1College of Resources and Environment, Hebei Polytechnic University, China.

2Key Laboratory of Mining Development and Safty of Hebei Province. China.

The continuous cropping system always led to nutrients disequilibrium in soil and inhibited plant

growth. In order to find a biological solution to deal with continuous cropping problems cow manure

vermicompost (CMV) was used into continuous cropping (over 10 years) tomato soil in laboratory

with consideration of ecology effect. The rate of CMV into soil were 0% (control, CK), 10%, 20%,

30% (in volume). The data showed that CMV accelerated plant growth rate. The dry weight of shoot

and root grown with CMV (10%, 20%, 30%) were more 31.6%, 15.3%, and 92.4% and 60.3%,

63.8%, and 112.1% than those of CK, respectively. The total N content in soil contained 20% CMV

after seedlings harvested decreased 1.9% than before planting. The total P content after planting in

soil with CMV (10%, 20%, 30%) reduced 93.2%, 49.2%, 45.8% than that of before planting,

respectively. On the country, the total P content in the CK after planting increased 14.5% than that of

before planting. It was to say that CMV stimulated plant absorption on P. Plant utilization for K in

three vermicompost levels increased, too. The content of total K in 10%, 20%, and 30%

vermicompost after planting reduced 45.4%, 40.9%, and 40.0% than that of before planting,

respectively. The difference of factors above were significant (P<0.05). The microbial populations in

soil with CMV were more than that of in CK. The fungi population in soil with vermicompost of

20% and 30% after planting was little or no change than that of before planting. It was indicated that

reasonable CMV application can control fungi population in continuous cropping soil. It was

concluded that CMV improved nutrients supply and maintained the microbial community stable in

continuous cropping tomato soil. It can be a modifying agent in continuous cropping soil.

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Session 7

Earthworms, Soil Contamination

and Bioremediation

233


5th to 10



Experiences with the earthworm avoidance test as a new tool for the

environmental risk assessment of individual chemicals and

contaminated soils

Roembke Joerg

1, Sousa Jose Paulo

2

1ECT Oekotoxikologie GmbH

2University of Coimbra, Portugal

The earthworm avoidance test using (mainly) the species Eisenia fetida or Eisenia andrei is a

relatively new ecotoxicological laboratory test which is characterized by its short duration and high

practicability (i.e. simple performance and low need of resources). Despite the fact that it is not

required in any regulatory context so far, it is becoming more and more popular as a screening tool in

lower tiers of site specific risk assessment schemes as well as for the evaluation of the effects of

chemicals, in particular pesticides. This popularity is partly caused by its simplicity, partly because it

has already been standardized by ISO (Guideline No. 17512-2, 2008). In this contribution, a brief

overview on the experiences made so far with this test is given. In particular, two issues will be

tackled: Firstly, when avoidance tests are used with site contaminated soils, the avoidance behaviour

of the test organisms can be influenced by soil properties which can act as confounding factors

creating highly biased results and misinterpretations. This can be problematic particularly when using

a reference soil (either an artificial or site soil) with different properties of the test soils. Using data of

a case study from Brazil, we will present the development and application of a GLM model to correct

for the influence of these confounding factors, thus decreasing the uncertainties associated to this

type of ecotoxicological data. Secondly, the toxicity of the worms observed in avoidance tests will be

compared with results gained in reproduction tests with the same soils. Here, we will use experiences

made in a case study from Germany in order to discuss the pro‘s and con‘s of the earthworm

avoidance test in order to evaluate the relevance of such test results for the environmental risk

assessment of contaminated soils.

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Oral – Session 7

Interaction of lead with earthworm secreted calcium carbonate granules

Hodson Mark E

1, Fraser Anne

1, Lambkin Denise

1, Lee Martin R.

2, Schofield Paul F.

3,

Mosselmans Fred4

1Dept. Soil Science, University of Reading

2Dept. Geographical and Geological Sciences, University of Glasgow

3Dept. Mineralogy, Natural History Museum

4Diamond Light Source

The influence of soil organisms on metal mobility and bioavailability is not currently fully

understood. We conducted experiments to determine whether the calcium carbonate granules

excreted by the earthworm Lumbricus terrestris L. could incorporate and immobilize lead in an

artificial soil amended with lead and calcium. Individual Lumbricus terrestris were each exposed to

300 g air dried OECD-style artificial soil (comprising 10 wt % peat, 20 wt % kaolinite and 70 %

quartz sand, all sieved to Average granule production rates of 0.39 +/- 0.04 mg calcite / earthworm /

day did not vary with soil lead concentration. The lead: calcium ratio in granules increased

significantly with that of the soil (r2 = 0.81, p = 0.015) with lead concentrations in granules reaching

1577 mg / kg. X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS) detected calcite

and aragonite in the granules. X-ray absorption spectroscopy indicated that some lead was present in

the granules as Pb3O4, possibly as a result of beam damage, cerrusite (PbCO3), hydrocerrusite

(2PbCO3.Pb(OH)2) and adsorbed to calcite and aragonite,. Despite their high lead concentrations the

likely mass of granules in soil of 3 to 30 g / m3 means that they are unlikely to account for significant

masses of lead in lead contaminated soil.

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Oral – Session 7

The effects of earthworm activities (Aporrectodea giardi) on the

distribution of geogenic arsenic and soil nutrients in the Jura Mountains

(Switzerland)

Le Bayon Claire

1, Matera V.

2, Kohler-Milleret R.

1, Degen C.

1, Gobat J.M.

1

1University of Neuchâtel - Laboratory Soil & Vegetation

2University of Neuchâtel - Laboratory of Geology

Environmental pollution due to arsenic (As) has been well studied and most investigations focused

on inorganic As coming from industrial areas. However, the natural rock-borne contamination of this

metalloid was underestimated, as well as the impact of arsenic on soil biota. Focusing on annelids,

only one study addressed the question of geogenic arsenic distribution in earthworm casts and arsenic

speciation in tissues. Moreover, to our knowledge, no research was conducted on the impact of

earthworms on soil arsenic behaviour. As a consequence, this study aimed to look at the influence of

earthworms on the mobilization (availability, transfer) of arsenic coming from a geogenic source. In

this context, we assumed that: 1- geogenic As should differ in biogenic structures (faeces, burrows)

compared to non-ingested soil, 2- the distribution and the bioavailability of geogenic As should be

modified due to earthworm bioturbation activities and gut processes, thus influencing the potential

As mobilization, 3- earthworms should favour the transfer of As in the soil profile through their

vertical movement, 4- earthworms may accumulate geogenic As in their own tissues, leading to a

modification of their biological traits. To test our hypotheses, microcosms in controlled conditions

were filled with soil containing 157 mg As kg-1 from a geogenic origin. The soil was collected from

a meadow situated at an altitude of 1330m in the Jura Mountains. As a result, our first assumption

was not verified: while earthworm surface-casts were enriched in carbon, nitrogen and available

phosphorus compared to non-ingested soil, no enrichment was found for arsenic. The second

hypothesis about the As-distribution showed no significant difference comparing soil texture in

surface-casts and non ingested soil. Arsenic predominated in the coarse sand fraction. Focusing on

arsenic potential mobilization, less As was released from earthworm surface-casts suggesting that the

humification of organic matter in the digestive tract of earthworms may help the formation of As-

humic/fulvic complexes. The third assumption supposed that earthworms should favour the transfer

of as in the soil profile. In our experiment using artificially enriched soil, almost 50 % of worms died

and some of the survivors entered into diapauses. As a consequence, the expected mixing between

the As-enriched soil section and the upper part of the column did not occur. Finally, we did not

demonstrate any geogenic arsenic effect on earthworm survival and growth. As a general conclusion,

it appeared that geogenic as slightly affects biological components and environmental aspects. These

findings are particularly positive taking into account the fact that most of the environments at an

altitude of 1330 m in the Jura Mountains present rocks coming through the soil, especially in

meadows that are regularly under the influence of the cattle.

236


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Oral – Session 7

Impact of earthworms on the remediation efficiency of contaminated soil

Sizmur Tom

1, Wingate Jeremy

2, Hutchings Tony

2, Hodson Mark E.

1

1University of Reading

2Forest Research

Pollutant pathways have been identified at the Parys Mountain mining site, Anglesey, UK. Ochre

ponds at the site are un-vegetated, with conditions inhibiting natural revegetation. The aim of this

study is to test the impact of compost and biochar, with or without earthworms, on the mobility of

metals and revegetation of the contaminated soil and see if the addition of earthworms compromises

remediation efforts. Contaminated ochre (1343 mg/kg Cu and 262 mg/kg Zn) was remediated with

compost and/or biochar in mesocosms in order to create a substrate suitable for plant growth.

Lumbricus terrestris (L.) were added to a treatment that was remediated with both biochar and

compost in order to determine the impact that they had on the mobility and availability of the metals

in the remediated soil. Mobility was determined by measuring the water soluble metals and

availability by measuring uptake into Common Bent (Agrostis capillaries). The combination of

biochar and compost decreased the mobility of Cu and Zn in the contaminated soil and increased the

pH from pH 2.7 to pH 6.7 with L. terrestris and 6.6 without. In the soil amended with both biochar

and compost the water soluble Cu was significantly decreased from 5.6 mg/kg to 0.2 mg/kg, a factor

of 248 and Zn was significantly decreased from 3.3 mg/kg to 0.05 mg/kg, a factor of 65. The addition

of L. terrestris however had no significant effect on the mobility of the metals in the remediated soil.

Common Bent did not germinate in the unamended control soil but both compost and biochar

increased the germination with the compost treatment producing the greatest growth (8 times greater

than the next highest treatment). The combination of compost, biochar and L. terrestris produced the

lowest concentrations of Cu (8.1 mg/kg) and Zn (35.7 mg/kg) in the aboveground biomass of the

grass; significantly lower than the compost treatment (15 mg/kg Cu and 126 mg/kg Zn) by a factor of

1.9 and 3.5 respectively. The addition of both biochar and compost, individually and together, was

effective at reducing the mobility and availability of metals and created a medium that is suitable for

revegetation and earthworm survival. The addition of L. terrestris did not re-mobilize previously

sequestered metals.

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Oral – Session 7

Suitability of Dendrodrilus rubidus and Lumbricus rubellus coelomocytes

for monitoring soil pollution: mini-review

Plytycz Barbara

1, Andre Jaqne

2, Morgan John

2

1Institute of Zoology, Jagiellonian University, Krakow, Poland,

2Cardiff School of Biosciences, Cardiff University, Cardiff, UK,

Abandoned Zn/Pb mine-sites and aerially-contaminated Ni-smelter sites in the UK are inhabited by

limited number of earthworm species, including the epigeics Dendrodrilus rubidus and Lumbricus

rubellus. Phylogenetic trees based on the COII mitochondrial gene of both species sampled from two

unpolluted reference sites and from four Pb/Zn-polluted sites in Wales revealed that although L.

rubellus comprises two distinct genetic lineages these are not differentially restricted to polluted or

unpolluted soils; our limited D. rubidus samples were genetically clustered into two separate clades

corresponding with polluted and reference sites, respectively. D. rubidus accumulated metallic soil

contaminants, especially Zn and Pb, much more efficiently than L. rubellus, and was found by ex

vivo counting after non-invasive extrusion from the coelomic cavity to possess a relatively higher

number of immune-competent cells (coelomocytes). These two lumbricid species belong to groups of

worms with contrasting coelomocyte communities and biochemistry: in L. rubellus coelomocytes are

almost exclusively amoebocytes; D. rubidus contain both the small non-granular amoebocytes and

large, granular, autofluorescent chloragocytes/eleocytes. Eleocytes contain fluorescence emitting

riboflavin stored in cytoplasmic vesicles. In L. rubellus, interpopulation differences in numbers of

amoebocytes and their pinocytic activity (established by flow cytometric analyses of neutral red

uptake) did not correlate with soil pollution intensity. The coelomocytes of D. rubidus from

unpolluted and metal-polluted soil samples were subjected to flow cytometry for determining the

percentage autofluorescent eleocytes, and to spectrofluorometry for riboflavin assays in coelomocyte

lysates. In D. rubidus, riboflavin content was a sensitive molecular biomarker of soil pollution:

riboflavin-derived autofluorescence was high only in the cells of worms inhabiting unpolluted soil

but was virtually extinguished in their counterparts inhabiting or transferred to field soils

contaminated with Zn/Pb or Ni/Cu. In conclusion, coelomocyte-connected parameters investigated so

far in L. rubellus can be modulated by soil quality perturbations, but they are not such sensitive and

robust biomarkers of soil metal pollution as is the easily measured riboflavin content of D. rubidus

coelomocytes. Acknowledgements: Supported by a grant (No PB3502/PO1/32) from the Ministry of

Science and Education (Poland).

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Oral – Session 7

The worm that turned? Inferring possible genetic adaptation of Lumbricus

rubellus (Hoffmeister) populations along a Ni-pollution gradient, and in

adjacent non-polluted geochemically contrasting soils

with microsatellite markers

Donnelly Robert

University of Glamorgan

Earthworms are particularly suitable for monitoring contaminated land because of their

ecophysiological characteristics, their role in modifying the physic-chemical properties of soils, and

their capacity to bio-accumulate contaminants, occasionally to concentrations exceeding those in the

native polluted soil. For these reasons, a range of biomarkers of sub-lethal stress have been

developed in certain species. Whilst these cover the spectrum of biological levels of organization, the

advent of contemporary ―omics‖ technologies have facilitated an intense interest in field-based

environmental diagnostics using earthworms. These studies are, however, potentially confounded by

site-specific micro-evolutionary events whereby resistant genotypes may not conform to the dose-

response predictions of laboratory toxicity tests. Moreover, the ecotoxicological implications of the

fact that several lumbricid species are differentiated into distinct genetic lineages have not yet been

fully assimilated. This study had two major aims: (i) to determine whether populations of the

cosmopolitan epigeic species, Lumbricus rubellus, are genetically differentiated along a gradient of

nickel contamination in the vicinity of the largest Ni-smelter in western Europe; and, (ii) whether the

genetic structure of L. rubellus populations native to adjacent calcareous and acidic non-polluted

soils are distinct. Gene flow was examined using microsatellite markers, the majority of which were

developed from expressed sequence tag sequences of L. rubellus, whilst two were amplified using

primers developed for a congeneric species. Preliminary results indicate a degree of genetic

heterogeneity to exist between populations of L. rubellus at some of the most heavily contaminated

sites along the Ni gradient. This could be indicative of adapted populations at these sites.

Contrastingly, little evidence of genetic heterogeneity was found between populations from

unpolluted soils of different pH, indicating that this edaphic parameter has little impact upon gene

flow. In conclusion, the presence of intraspecific genetic heterogeneity at contaminated sites could

present a problem for ecotoxicological studies because such studies implicitly assume L. rubellus to

be genetically homogenous, and thus display variations in genetically-determined traits reflecting

site-specific pollution intensities. Acknowledgement: We are grateful to the Levehulme Trust for

funding this project (Ref. F/00407/AI).

239


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Oral – Session 7

Earthworms, are a means to accelerate removal of hydrocarbons from

soil? A review

Contreras Ramos Silvia Maribel

1, Alvarez Bernal Dioselina

2, Dendooven Luc

3

1Environmental Technology Unit, Center for Research and Assistance in Technology and Design of the Jalisco

state, A.C. (Ciatej), Mexico 2Interdisciplinary Research Center for the Integral Regional Development (CIIDIR-Michoacán)- I.P.N. Mexico

3Department of soil ecology, Centro de Investigación y Estudios Avanzados del I.P.N. (Cinvestav), Mexico

Contamination of soil with hydrocarbons occurs frequently in Mexico, one of the most important

petroleum producers in the world. Restoring contaminated soils is costly and time consuming.

Earthworms might help to accelerate the removal of hydrocarbons as they burrow through soil,

feeding on the organic matter, improving soil structure and aeration. Polycyclic Aromatic

Hydrocarbons (PAHs) are of the most recalcitrant hydrocarbons that can contaminate the

environment and can thus serve as models to study the factors that control the removal of petroleum

from soil. We report on the effect of earthworms on the removal of phenanthrene, anthracene and

benzo(a)pyrene from soil and dynamics of C and N. It was found that earthworms have the capacity

to accelerate the removal of PAHs from soil and increase the mineral N content. However, their use

in a contaminated field will require a high density of the earthworms, which might be expensive and

the provision of sufficient organic material as feed while a high soil water content must be

maintained to stimulate burrowing.

240


5th to 10


Oral – Session 7

Genetic diversity of Lumbricus rubellus in unpolluted and polluted habitats

Pinto Juma Gabriela A.

1, Donnelly Robert

2, Bundy Jacob G.

3, Bruford Michael W.

1,

Morgan John1

1School of Biosciences, Cardiff University

2Faculty of Health, Sport and Science, University of Glamorgan

3Biomolecular Medicine, Imperial College

Earthworms have been extensively used in terrestrial ecotoxicology due to their well-known

ecological engineering functions and ecophysiological characteristics. Micro-evolutionary events,

such as local population adaptation to elevated concentrations of soil contaminants may, however,

confound the deployment of biomarkers in field-based ecotoxicological assessments. In order to

assess the utility of biomarker data we evaluated and compared genomic variation and dispersal in

populations of the sentinel earthworm Lumbricus rubellus, in geochemically diverse unpolluted and

polluted habitats. Specimens of L. rubellus were collected from sites along a Nickel (Ni) gradient

downwind and upwind from a working Ni smelter at Clydach (Wales, UK), and also along transects

centred on two unpolluted reference sites with contrasting soil types (Dinas Powys and Rudry

Common). The chemical composition of soil samples from each site was analyzed comprehensively

for 23 elements by ICP-OS, and all earthworms collected (30 individuals from c. 10 stations per site)

were genotyped using mitochondrial cytochrome oxidase II (COII) sequences. 210 specimens from

twelve selected sites were further screened at the whole genome level using Amplified Fragment

Length Polymorphism (AFLP). These analyses resolved L. rubellus into at least two mitochondrial

lineages (‗A‘ and ‗B‘) with an average p distance of 11% between them. A differential distribution of

lineages along the Ni-pollution gradient suggests that each lineage may express different levels of Ni

tolerance. Ni contaminated sites show the same level of genetic diversity as non-polluted sites,

suggesting that the populations closer to the contamination source may represent ―demographic

sinks‖, sustained by the recruitment of non-adapted individuals from adjacent less contaminated land,

which may possibly adapt to their new relatively stressful surroundings through phenotypic

plasticity. Our observations did not support the notion of genetic erosion in the vicinity of a pollution

point source. The role of this soil-dwelling species as a sentinel organism in laboratory- and

(especially) field-based ecotoxicological studies should assimilate the knowledge that it is

differentiated into at least two deeply distinct genetic lineages, each lineage possibly comprising

ecotypes of significantly different metal sensitivity. Acknowlegement: This study was supported by a

research grant (F/00407/AI) awarded by The Leverhulme Trust.

241


5th to 10


Oral – Session 7

Molecular toxicology of cadmium on earthworms in low polluted soil

Mo Xiaohui, Qiao Yuhui, Li Yang, Sun Zhenjun


An experiment was carried out in order to accurately reflect the actual exposure or the potential

biological toxicity of heavy metals to earthworm in the polluted soil under low-dose cadmium

concentrations. In this study, changes of physiological related genes expression of earthworms were

detected using real time PCR under long-time of 70 days‘ pollution. The physiological genes selected

were MTs, Annetocin, Calreticulin and Anti-microbial peptide and two types of soil; natural soil with

Cd content of 0 and 1.46 mg/kg (pollution-free soil and polluted soil) and artificial soil with Cd

content: 0, 1, 5, 10, 25, 50 mg/kg, were studied in the experiment. The results showed that the

expression of metallothionein gene (MTs) increased significantly with the increase of soil cadmium

concentration. For artificial soil treated earthworms, when the cadmium concentrations come up to

50 mg/kg, the MTs expression was 12.68 times of that of the CK. It showed a significant positive

correlation of dose-response relationship. For natural soil treated ones, MTs expression of ―polluted

soil‖ groups was 2.4 times of that of the ―pollution-free soil‖. The expression of earthworms‘

production hormone gene (Annetocin) was gradually reduced with the increase of the soil cadmium

concentration. For artificial soil treated earthworms, the Annetocin gene expression of ―50mg/kg‖

groups was 0.243 time of the CK groups. It manifested a significant negative correlation of dose-

response relationship. In the meantime; expression of ―Polluted soil‖ treated earthworms was 0.214

time of ―pollution-free soil‖ treated ones. There was no significant correlation between the soil

cadmium concentration and the Calreticulin expression of the treated earthworms. For artificial soil

treated earthworms, the Calreticulin expression level of ―1 mg/kg‖ groups was the highest. For

natural soil treated ones, Calreticulin expression of ―polluted soil‖ groups was also higher than that of

the ―pollution-free soil‖. Gene expression of Anti-microbial peptide was not changed obviously for

artificial soil treated earthworms under low-dose cadmium. The existence of a higher dose of

cadmium (10 mg/kg) may increase the expression level. However, if the dose was too high, the

existence of cadmium may also have inhibiting effect on the genes expression of Anti-microbial

peptide. For natural soil treated ones, there was no significant difference between the ―polluted soil‖

groups and the ―pollution-free soil‖ groups.

242


5th to 10


Oral – Session 7

Metabolomics in earthworm biology

Bundy Jake

Imperial College London

Metabolomics — the untargeted profiling of small-molecule metabolites — is a powerful approach to

measuring subtle phenotypic differences. We have been applying metabolomics to a number of

different problems in earthworm biology, including understanding responses to toxic chemicals and

other environmental stresses. In addition, it can be used to study genotype/phenotype differences, and

how these interact with environmental factors. Here we will present data from a number of studies

using nuclear magnetic resonance (NMR) spectroscopy as an analytical tool, including controlled

lab-based exposures to single toxic chemicals, field studies at contaminated sites, and assessing

functional differences between populations. The results demonstrate some of the potential advantages

of the NMR metabolomic approach: for instance, chemicals with different toxic modes of action lead

to differing metabolite responses, implying that potentially soil contaminant effects could be

identified by ‗fingerprinting‘. In addition, an example is given where the observed metabolite

responses lead to clear and directly testable biochemical hypotheses. Extending this to the field level,

we could identify a toxic metal (zinc) as the most probable cause of actual functional effects, even

across sites with very different underlying soil type, vegetation, etc. Finally, we show how

metabolomic phenotyping can be used to help the interpretation of genotype information in natural

populations.

243


5th to 10


Oral – Session 7

Mixture interactions and mechanisms of joint effects of stressors in

earthworms exposed to stressor combinations

Spurgeon David

1, Svendsen Claus

1, Kille Peter

2, Morgan John

2

1Centre for Ecology and Hydrology

2University of Cardiff

In soil ecosystems, earthworms are exposed to combinations of stressor including toxic chemicals,

oxygen depletion, climate extremes and pathogens. Describing and understanding the nature of these

combined effects requires the integration of statistical modelling approaches with methods that

elucidate the mechanisms responsible for joint effects. Established concepts used in mixtures

pharmacology (notably the concentration addition and independent action models) can be used to

predict the effects of both mixtures of chemicals and combinations of chemicals with other stress

factors. In many cases, such as mixtures of neonicotinamide pesticides, metals and PAHs when

exposed in increasingly dry soils, these standard models have been found to effectively describe joint

effect. In other cases, however, the standard models fail to account for actual effects; responses being

either synergistic or antagonistic. In cases where the available models fail, the assumption must be

that interactions between the chemicals and/or other stressor mean that the conceptual basis (that of

strict and independent additivity) of the models are not met. Where such interactions occur,

approaches to address the temporal nature of exposure (e.g. toxicokinetics) and/or mode of action can

be used to investigate the basis of the interactions in earthworms. Temporal approaches to address

exposure include toxicokinetic models based on simple and more complex compartment models that

can be utilized in earthworms. For mode of action assessment, the emergence of functional genetic

tools has allowed a deeper investigation of the common and unique transcriptional responses that

delineate the common response and distinct mechanistic effects of different chemicals in a mixture.

244


5th to 10



Bioaccumulation of cypermethrin in earthworms Eisenia andrei from

different soils

Alves de Sousa Ana Paula, De Andréa Mara Mercedes

1Instituto Biológico, Brasil

The pyrethroid insecticide cypermethrin has been widely used in agriculture and public health

campaigns due to it is efficiency in controlling insects, short persistence in the environment and low

toxicity to birds and mammals. However, a large portion of the applications of cypermethrin may

leave toxic residue in the soil and, organisms such as earthworms, may be exposed to these

contaminants in soil, which are absorbed by earthworms or bioaccumulated in their tissues. As

earthworms are at the base of the food chain serving as food for other organisms, studies on

bioaccumulation of pesticides in these organisms may indicate the potential for contamination along

the food chains. This study determined the bioaccumulation factors -FBA 14

C-cypermethrin on

earthworm Eisenia andrei from three soil types: Typic Hapludox (LV), Mollic Hapludalf (PV) and

Typic Humaquept (GM). Four replicates of 100 g of each soil type were treated with 0.05 mg and

0.056 kBq of 14

C-cypermethrin g-1

soil. The controls received 400 mL of acetone. After solvent

evaporation, five earthworm specimens with clitellum and more than 300 mg each were placed in

each sample. The study was conducted in a temperature-controlled chamber at 22 °C ± 2 °C and

continuous light for 14 days. At the end of 14 days, 10 g of each type of soil were extracted with

methanol by mechanical agitation. The earthworms in each chamber were sacrificed by freezing at -

18 °C for 20 minutes and 14

C-cypermethrin was extracted from their tissues with methanol for 30

cycles of 15 seconds and 160 W of microwave energy. The bioaccumulation factors (FBA) was

calculated by the following formula: Ctm = FBA / Cs, where Ctm: concentration of [14

C] in the

tissues of earthworms (kBq g-1

) and Cs: Concentration of [14

C] in soil (kBq g-1

). The values of FBA

showed that cypermethrin was bioaccumulated by earthworms, regardless of soil type; therefore the

FBA was 3.73 ± 0.37 in LV, 3.52 ± 1.09 PV and 2.11 ± 0.73 in GM. It was verified less FBA in soil

with high organic content (GM), which is the factor that usually determines the pesticide residues-

bounds formation, and therefore less available to organisms. However, it was found that

cypermethrin is bioaccumulated and this contamination can occur throughout the food chain from

earthworms.

245


5th to 10



Reducing the toxicity of bone meal to earthworms so that it can be used to

remediate metal-contaminated soil

Atuah Laura,

Hodson Mark E.

Dept. Soil Science, University of Reading

Bone meal is an organically derived source of phosphorus (P) that could potentially be used as a P

amendment to remediate metal-contaminated soil. Despite successful laboratory trials using a variety

of sources of P to immobilize metals, there are relatively few studies that investigate impacts of P

amendments on earthworms or other soil biota. Toxicity tests indicate that bone meal could have a

significant impact on earthworm mortality. It is hypothesized that the organic component of bone

meal is the source of toxicity. The aim of this study was to establish whether bone meal toxicity

could be reduced by simple, cost-effective treatments. Standard OECD methodology was adapted for

all toxicity assessments. Individual Eisenia fetida were exposed to 50 g of meal, no treatments

showed 100% mortality. Experiment 2: The time between bone meal amendment and earthworm

introduction was varied for soils amended with bone meal at a concentration of 3500 mg/P kg, i.e.

approximately the LC50 for untreated bone meal. Earthworms were introduced immediately after

amendment and then 7, 14, 21, 28 and 35 days after amendment. Delaying the introduction of

earthworms into bone meal amended soil reduced mortality rates. Experiment 3: Soils were amended

with both green waste compost (GWC) at rates of 5 to 25 % by weight and bone meal at 3500 mg P /

kg. Addition of GWC reduced earthworm mortality though there was no increase in this reduction

with increasing amounts of GWC. Our results are consistent with bone meal toxicity being associated

with release of organic components to the soil from the bone meal. The toxicity is transient and can

be reduced by simple treatments.

246


5th to 10



Bioavailability and Ecotoxicity of altered TiO2 nanocomposite on the

earthworm, Eisenia fetida

Bigorgne Emilie

1, Foucaud

1, Lapied Emmanuel

2, Joner E.

2, Chaurand

3, Labille Jérôme

3,

Rose3, Exbrayat Jean-Marie

4, Rodius

1, Nahmani Johanne

1

1UMR 7146 CNRS

2Norwegian University of Life Sciences

3CEREGE

4Université de Lyon

The quantity of nanomaterials produced annually is in constant increase. This trend may induce an

increase of the release of nano by-products in air, water, soils, and therefore in contact with

organisms. Up today, ecotoxicological risks associated with nanoresidues from alteration and aging

of nanomaterials are poorly documented. The aims of the present study were firstly to identify and

characterize altered TiO2 nanocomposite suspensions derived from particles usually included in

sunscreen cream, and secondly to evaluate their potential toxicity on the earthworm, Eisenia fetida.

In order to simulate the alteration of the nanocomposite, an accelerated aging process was performed

by mixing TiO2 nanocomposite with water during 48h. Then, a stable solution of TiO2 rich

nanoresidues was obtained, and chemically and physically characterized. Earthworms were exposed

to altered nanocomposite solutions at 4 concentrations (0, 1, 5 and 25 mg.L-1

) during 24h in the dark.

The different biological parameters included survival, bioavailability, immunotoxicity and

detoxication system for which metallothionein, superoxide dismutase and pi-class Glutathione-S-

tranferase mRNA expressions on earthworm‘s gut were measured by real-time PCR and cocoon

production. Results showed that the altered TiO2 nanocomposite had no effect on survival rate and

did not modify gene expression in the earthworm‘s gut. At contrast, cocoon production seems to be

reduced at all conditions. Observations of titanium absorption with X-ray fluorescence microscopy

show titanium embedded with residual soil ingested by earthworm‘s gut but not in the tissue. Results

on total metal concentrations (Al/Ti) in tissues show a significant increase of titanium in earthworm‘s

tissue. immunotoxicity (phagocytosis and cellular viability).

247


5th to 10



Pontoscolex corethrurus (Müller, 1857) and Eisenia andrei, Bouché, 1972 as

bioindicators of soil contaminated by pesticides

Cristhy Buch Andressa

1, Brown George

2

1Universidade Federal do Paraná

2Embrapa - Florestas

The indiscriminate and excessive use of pesticides has been constantly increasing due to the demand

for greater agricultural production. This intense use of soil impacts the environment and soil biota.

To indicate the extent to which these chemicals are harmful and how and where their effects occur,

you can make use of ecotoxicological tests with earthworms. These animals are essential for the

maintenance of chemical and biological processes in the soil and are useful to evaluate impacts of

pesticides through behavioural, acute and chronic tests. Some of these tests were standardized by ISO

(International standardization organization) using the earthworms Eisenia fetida and Eisenia andrei,

both native to temperate climate. However, these species may be of little relevance for

ecotoxicological seraphic studies, because they live in the soil litter (epigamic), and consume mainly

fresh organic matter. The species Pontoscolex corethrurus, native to tropical regions, can be an

alternative to an ecotoxicological most ecologically important species, because it is a true soil living

organism and consume soil organic matter (endogeic). Little is known about their sensitivity to

pesticides though. Therefore, in the present study, the sensitivity of E. andrei and P. corethrurus

when exposed to three pesticides often used in fruit and grain in Brazil - carbendazim, carbofuran

and glyphosate – were tested in a behavioural test ISO 17512-1, 2007 and an acute mortality test,

ISO 11268-1, 1993. The results showed that carbendazim and carbofuran were toxic to both species.

For carbendazin, E. andrei showed EC50 of 76.12 mg i.a. kg-1

and LC50 of 19.74 mg i.a. kg-1

, while

for P. corethrurus, EC50 of 65.81 and LC50 of 15.32 mg i.a. kg-1

. For carbofuran, E. andrei showed

EC50 of 9.74 and LC50 of 13.50 mg i.a. kg-1

while for P. corethrurus EC50 of 7.26 and LC50 of 9.28

mg i.a. kg-1

. Glyphosate, on the other hand, showed no toxic effects at the tested concentrations for

both species. The EC50 obtained for both species were very similar, 44.52 mg i.a. kg-1

for P.

corethrurus, and 45.52 mg i.a. kg-1

for E. andrei. The sensitivity of P. corethrurus seems to be

statistically similar when compared with the standard species E. andrei for the pesticides evaluated.

248


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To reveal the relationship of earthworm and UVC: from a platform for

assessment of dynamic DNA damage/repair capacity in fresh-prepared

earthworm cells

Da-Tian Bau

1, Chung-Hsing Wang

1, Chia-Wen Tsai

1, Ru-Yin Tsai

1, Cooper Edwin L.

2,

Fuu-Jen Tsai3, Chang-Hai Tsai

4, Chi-Yang Huang

4

1Terry Fox Cancer Research Lab, China Medical University Hospital, Taichung, Taiwan

2Laboratory of Comparative Neuroimmunology Department of Neurobiology University of California, Los

Angeles; California, USA 3Graduate Institute of Basic Medical Science, Department of Chinese Medical Science, China Medical

University and Hospital, Taichung 404, Taiwan 4Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan

DNA is a stable molecule which is highly sensitive to all kinds of DNA damage and even a subtle

alteration can be detected via specific assays. Therefore, the assessment of DNA damage is

considered important in toxicity testing. However, few studies on the assessment of genotoxic risks

of DNA damage agent for earthworms due to the difficulties in quantifying exposure levels,

bioavailability, and effects. The specific aim of this study is to determine dynamic DNA damage and

repair process in earthworms exposure to different DNA damage agent (including: UVC, 4NQO,

BPDE, cisplatin, MMC, MMS, H2O2, SNP, or SIN-1

), and to reveal the relationship between

earthworm and UVC.

249


5th to 10



Molecular mechanism associated with heavy metal tolerance in three

epigeic earthworms

Dinesh M.S.

1, Geetha K.S.

1, Kale Radha D.

2

1Department of Biotechnology, PES Institute of Technology

2Center for Scientific Research and Advanced Learning, Mount Carmel College

Tolerance to heavy metals is the salient feature exhibited by earthworms. Earthworms have the

mechanism for accumulating heavy metals in the chloragogenous cells that prevent them to creep

into blood stream with the help of metal binding protein namely metallothionein. Metallothionein is

secreted in the earthworm body in response to heavy metal exposure as a detoxification mechanism.

The present study is aimed to find out the limit of tolerance to heavy metals and associated molecular

mechanism exhibited by three species of epigeic earthworms, namely, Eudrilus eugeniae, Perionyx

excavatus and Eisenia fetida. These earthworms were exposed to Lead (Pb) and Cadmium (Cd) at the

concentration range of 1mg to 10 mg/kg of partially decomposed feed. After 30 days of composting

period, the heavy metal levels in the earthworm body were analyzed using atomic absorption

spectrophotometer. The results revealed that the tolerance to lead among the species was 2 mg/kg for

Eudrilus eugeniae, 3mg/kg for Perionyx excavatus and 6mg/kg of feed for Eisenia fetida. The

accumulation levels in their bodies were found to be 112, 225 and 350 ppm respectively. The

tolerance levels for Cadmium in the same order of species were 4 mg/kg, 6 mg/kg and 8 mg/kg of

feed with accumulation levels in the body ranges from 160, 252 and 390 ppm. The amount of

metallothionein recorded on exposure of these earthworms was in the order 40, 60 and 120 µg/g of

body tissue for both Lead and Cadmium for Eudrilus eugeniae, Perionyx excavatus and Eisenia

fetida. The metallothionein was purified using affinity column chromatography with sephadex G200.

Purified protein is subjected to N & C terminal sequencing in a Shimadzu sequencer by automated

Edman degradation method. A sequence of 9 amino acids namely Ala-Gly-Ser-Tyr-Thr-Pro-PheAla-

Val-Leu-Isoleu was found to be responsible for metal binding property of metallothionein that is

stored in the chloragogenous tissue. The present study revealed that Eisenia fetida exhibited

maximum tolerance to heavy metals followed by Perionyx excavatus and Eudrilus eugeniae. All the

three species together can be used for bioremediation of Pb and Cd contaminated soil since they have

tolerance.

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Changes in forms of Lead and Manganese with passage through the

intestinal tract of Pontoscolex corethrurus (Oligochaeta: Annelida)

Duarte Ana Paula

1, De Freitas Melo Vander

2, Pauletti Volnei

2, Brown George

3

1Programa de Pós-Graduação em Ciência do Solo, Universidade Federal do Paraná, Setor de Ciências Agrárias,

Rua dos funcionários, 1540, 80.035-050, Curitiba, PR, Brazil 2Universidade Federal do Paraná, Setor de Ciências Agrárias, Departamento de Solos, Rua dos funcionários,

1540, 80.035-050, Curitiba, PR, Brazil 3Embrapa Florestas, Estrada da Ribeira km. 111, C.P. 319, Colombo-PR, 83411-000, Brazil

Mining and industrialization are major sources of environmental pollution and soil contamination,

thus affecting soil food webs. Earthworms are sensitive to soil contaminants and can be used as

bioindicators of soil pollution with heavy metals. On the other hand, their bioturbating activities in

soils can alter soil chemistry and the availability of various elements. However, little is known

regarding the capacity of tropical earthworms to alter the forms of heavy metals in soils. Therefore,

the present study was undertaken to evaluate the effect of the passage of soil containing different

amounts of heavy metals through the intestinal tract of Pontoscolex corethrurus on the forms of lead

and manganese. The transformation of labile forms to more stable forms was assessed using the

sequential extraction of Pb and Mn on five soils from a lead mining site in Adrianópolis country,

Parana State, Brazil. Five adult worms were incubated in each plastic container with 300 cm3 of each

soil type (n=3 replicates), maintained at 70% field capacity and room temperature until enough

castings were produced for the analyses. Castings and control soil were collected every two days

using tweezers and the analyses were performed on uningested soil (control) and casts. Statistical

differences between uningested soil and castings were tested using ANOVA and Tukey-tests.

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Synergic Effect of Heavy Metals in Automobile Waste on the Earthworm

Eudrilus eugeniae

Gobi

Muthukaruppan, Gunasekaran Paramasamy

Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai 21, Tamilnadu,

India

In most of the developing countries including India, soil pollution due to industrial activities and

urbanization is of great concern, affecting adversely soil organisms especially earthworms. The

epigeic earthworm Eudrilus eugeniae was exposed to 100%, 75%, 50%, 30%, 25%, 15% and 5% of

automobile service station waste mud. Each concentration level was tested with three replicates using

10 animals. In flow-through experiments life history parameters such as biomass, clitellum

development, and cocoon production were measured. After exposing the earthworm to the service

station waste mud, mortality was observed in the 100%, 75%, 50% and 35% concentrations. So,

further experiments were conducted within 25%, 15% and 5% levels. At the end of the experiment,

the control group had a mean biomass of 2.814 ± 0.235 mg and, in the 25%, 15% and 5% exposed

group, the mean biomass 2.531 ± 0.094 mg, 2.712 ± 0.123 mg and 2.926 ± 0.276 mg, respectively.

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Apoptotic activity as a toxicity response in the earthworm Eisenia fetida

(Oligochaeta) after exposure to TiO2 nanoparticles

Lapied Emmanuel

1, Bigorgne Emilie

2, Moudilou Elara

3, Labille Jérôme

4,

Exbrayat Jean-Marie3, Oughton Deborah H.

5, Joner E.

1, Nahmani Johanne

2

1Bioforsk, Soil and Environment, Fredrik A. Dahls vei 20, N-1432 Ås, Norway

2Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes (LIEBE), UFR Sciences

Fondamentales et Appliquées. Université Paul Verlaine, Campus Bridoux, Rue du Général Delestraint, 57070

Metz, France 3Laboratoire de Biologie Générale, UCL, Laboratoire de Reproduction et Développement Comparé, EPHE,

UMRS 449, Université de Lyon, 25 rue du Plat, F-69288 Lyon Cedex 02, France 4Centre Européen de Recherche et d‘Enseignement des Géosciences de l‘Environnement (CEREGE). Europole

Méditerranéen de l'Arbois, BP 80, 13545 Aix en Provence cedex 04, France 5Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-

1432 Ås, Norway

In parallel to a dramatic increasing production and use of engineered nanoparticles (ENPs) in

nanotechnology applications, investigations on potential physiological and ecotoxicological effects

are currently emerging. However, still few it is known about potential impacts of nanoparticle

releases on soil macrofauna, and in particular on earthworms, a key component in soil structuration

and decomposition processes. Beyond the complex interactions between ENPs and the soil matrix,

this is partly due to the lack of suitable toxicological endpoints in organisms exposed to ENPs. In this

work, we exposed the earthworm Eisenia fetida to various concentrations (1-25 mg l-1

) TiO2

nanoparticles in water solution under three different pH values (pH 5.5 to pH 6.5). After exposure,

we measured the programmed cell death activity in various tissues using two staining techniques to

determine apoptosis: TUNEL and Apostain. A dose-dependent response was detected with respect to

apoptotic activity in a range of tissues, mainly cuticule, intestinal epithelium and chloragogenous

matrix, at the most acute concentration. Apoptosis thus seems to be a sensitive endpoint for detection

of adverse effects of TiO2 nanoparticles, and potentially a powerful tool for quantifying

environmental hazards posed by ENPs.

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Mode of exposure and nanoparticle size determine toxicity of silver

nanoparticles measured as apoptosis in the earthworm Lumbricus terrestris

Lapied Emmanuel

1, Moudilou Elara

2, Exbrayat Jean-Marie

2, Oughton Deborah H.

3,

Joner E. 1

1Bioforsk Soil and Environment, Fredrik Dahls vei 20, N-1432 Ås, Norway

2Laboratoire de Biologie Générale, UCL, Laboratoire de Reproduction et Développement Comparé, EPHE,

UMRS 449, Université de Lyon, 25 rue du Plat, F-69288 Lyon Cedex 02, France 3Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-

1432 Ås, Norway

In terrestrial ecotoxicology there is a serious lack of data for potential hazards posed by engineered

nanoparticles (ENPs). This is partly due to complex interactions between ENPs and the soil matrix,

but also to the lack of suitable toxicological endpoints in organisms that are exposed to ENPs in a

relevant manner. Earthworms are recognized as key organisms in terrestrial ecosystems, but so far

only physiological endpoints of low sensitivity have been used in ecotoxicity studies with ENPs. We

exposed the earthworm Lumbricus terrestris to different concentrations (1-100 mg l-1

) silver

nanoparticles of two contrasting sizes under three different conditions: in water, in food and in soil.

After exposure, we measured the impact on apoptosis in different tissues using two staining

techniques: TUNEL and Apostain. A dose-dependent response was detected with respect to apoptotic

activity in a range of tissues both at acute and sub-lethal concentrations (down to 4 mg kg soil-1

).

Comparing exposure in water and soil showed that reduced bioavailability in soil is reflected in the

apoptotic response. Apoptosis thus seems to be a sensitive endpoint for detection of adverse effects

of silver nanoparticles, and potentially a powerful tool for quantifying environmental hazards posed

by ENPs.

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Remediation of Cadmium Contaminated Soil and its affective factors by

earthworm casts

Li Yang, Qiao Yuhui, Sun Zhenjun, Mo Xiaohui

China Agricultural University

With the development of industry and agriculture, the contamination of soils and crops by heavy metals

has became more and more serious, especially for Cadmium deposited in soils, which is very harmful to

plants and human beings. In this paper, soil culture experiment was carried out to study the remediation

of cadmium contaminated soil by earthworm casts. In this experiment, different proportions

(0%，10%，25%，40%，50%) of earthworm casts were put into those soils which were polluted by

different concentrations of Cd (1.0, 5.0 and 20mg/kg). Different fractions of Cd in soils were determined

by BCR （Community Bureau of Reference) method in order to verify the inactive function of

earthworm casts and possible affective factors like pH, Cation exchange capacity and Humus acid in

those treated soils were determined by traditional methods in order to find out the crucial factors for

remediation function of earthworm casts. And then, pot experiment and analysis of crucial factors were

carried out to validate the inactive effect of earthworm casts and do some elementary studies on the

mechanism of amendment by earthworm casts. The mainly results from this paper showed as follows: (1)

The addition of earthworm casts could significantly (p<0.05) reduced the Ex-Cd and Oxides-Cd contents

extracted by HAc and NH2OH·HCl in soil polluted by low content of Cd (1.0 mg/kg), and the more

earthworm casts added the lower bioavailability of Cadmium was. Soils contaminated by 5.0, 20mg/kg

Cd had the same situation as the soil polluted by 1.0 mg/kg Cd, but the interaction is not more significant

than that in lower polluted soils. (2) pH value in soils polluted by Cd (1.0, 5.0, 20mg/kg) located between

7.210 and 7.473 after the addition of earthworm casts. Compared with the control, CEC and humus acid

had significantly（p<0.05）increased by about 30% and 200% respectively by adding 10% earthworm

casts. In addition, CEC and humus acid had significant（p<0.05）and negative correlations with the

content of Ex-Cd in soils. Therefore, CEC and humus acid were two crucial affective factors influencing

the immobilization of Cd by earthworm casts. (3) The addition of earthworm casts in 5.0 and 20mg/kg Cd

polluted soils could promote the growth of Chinese cabbage significantly (p<0.05). The biomass of

Chinese cabbage treated by earthworm casts was 2～3 times than that in control. In addition, no matter

which kind of Chinese cabbage, the content of total Cd absorbed by plant tissues decreased by about

50%. Cd concentration decrease from 9.386mg/kg to 4.506mg/kg in high accumulated Chinese cabbage

and from 3.356mg/kg to 1.583mg/kg in low accumulated Chinese cabbage in 5.0 mg/kg Cd polluted soils

after adding earthworm cast. Therefore, Cadmium in soils were immobilized significantly (p<0.05) by

earthworm casts.

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Biochemical responses of the earthworm Eisenia andrei exposed to

contaminated natural soil, effects of glyphosate

Marcano Leida, Hernández Juany, Marcano Elena, Cortesía Adriana, Polo Aldo

Universidad de Oriente, Venezuela. Lab. de Bioquímica y Ecotoxicología. Postgrado de Biología Aplicada

We investigated the effects of a commercial formulation of glyphosate (N-

(phosphonomethyl)glycine) herbicide, at different exposure times on biochemical responses of

Eisenia andrei to (1) elucidate the mechanisms of action of glyphosate and (2) investigate the

potential for using these responses as biomarkers to monitoring pesticide contaminated soils. Thus,

earthworms were exposed a glyphosate (100 g/kg natural soil) for 7 and 21 d using the standard soil

test method. After exposure period, a group of earthworms were depurated for 30 days. Reduced

glutathione (GSH) concentrations and activities of glutathione peroxidase (GPx) and glutathione-S-

transferase (GST) were measured in muscle tissue as antioxidant defences. Malondialdehyde (MDA)

tissue levels were determined as biomarker of lipoperoxidation in cellular membranes. Earthworms

exposed to glyphosate showed increased MDA production in muscle tissue at both exposure times.

Similarly, GPx and GST activities increased during of exposure to herbicide. GPx and MDA were

reduced in earthworms depurated. These results showed that glyphosate and its other chemical

compounds altered cellular antioxidant defences and induced oxidative stress in E. andrei. Oxidative

damage was reverted in depuration phase indicating that the effects observed in exposure period were

due to experimental treatment. The biochemical parameters measured can be used as herbicide

toxicity indicators considering environmentally relevant concentration.

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Mapping the distribution of Ni and Cu in surface soils and biota

(lumbricid earthworms and a grass) in the vicinity of a working smelter

Morgan John, Ingram K., Rowson M., Davies M.S., O'Reilly M., Brabham P.

Cardiff University

Ni is an essential trace element whose bioavailability, and consequent toxicity toward terrestrial

plants and animals, is both dependent on edaphic factors (notably pH) and on the ecophysiology of

receptor organisms. Ni is a recognized genotoxic metal. Some plants are efficient Ni accumulators,

but there is no evidence that any soil-dwelling invertebrate has a similar trait. Earthworms are

acknowledged macro-accumulators of several different essential and non-essential metals, and yet

regression analysis of the rather meagre body of literature data indicates that earthworm [Ni]: soil

[Ni] relationship has a modest negative slope. Thus, there is considerable uncertainty about Ni

bioaccumulation by an invertebrate taxon whose ecological engineering functions has conferred upon

it a prominent status in terrestrial ecotoxicology. The uncertainties concerning the Ni relationships of

earthworms arise mainly from the limited number of field studies that have been undertaken and the

limited range of soil Ni concentrations that have been examined. The soils around the long-

established (1902) and still working INCO Ni smelter in the Swansea Valley (UK), the largest in

Western Europe, therefore presents an excellent biomonitoring opportunity. We have analyzed and

mapped (with ‗Surfer‘ software) the spatial distribution of Ni in soil (conc. HNO3 digests and or 1M

ammonium acetate extractions), lumbricid earthworms, and the grass Agrostis capillaris across 36

locations up- and down-wind of the point source. Soils adjacent (~100 m) to the smelter stack were

found to contain c.3000 µg Ni/g dry mass (compared with Soil Guideline Value for residential soils

of 75 µg Ni/g). The slope of the downwind Ni gradient was steep and the edaphic heterogeneity (e.g.

soil pH ranging from 3.91 to 7.69) was noteworthy. Mapping revealed an unsuspected Ni-pollution

‗hotspot‘ coinciding with an historical loading area which bears no current physical remnants. The Ni

biconcentration factor (earthworm [Ni : soil ‗total‘ [Ni]) was found to be exceptionally low, with this

transition metal behaving akin to another, Cu. Acknowledgement: This study was completed during

the tenure of a research grant (F0 0407/AI) awarded by The Leverhulme Trust to AJM.

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Micro-focus X-Ray Absorption Spectroscopy of Pb and Zn in different cell

types in two earthworm species

(Dendrodrilus rubidus and Lumbricus rubellus)

Morgan John

1, Mosselmans Fred

2, Gunning P.,

3, Kille Peter

1

1Cardiff University

2Diamond Light Source Ltd

3Smith & Nephew

Station I18 at the Diamond Light Source Ltd (UK) has established an X-Ray Absorption

Spectroscopy facility capable of performing metal speciation by the micro-XAFS (Extended X-ray

Absorption Fine Structure) mode in heterogeneous structures at the ?m-level of spatial resolution.

This capability has been exploited in the present study to characterize the atomic environments

surrounding sequestered Pb and Zn centres, respectively, in the chloragocytes, intestinal epithelia,

and nephridial cells of two epigeic earthworms inhabiting a heavily contaminated derelict Pb/Zn

mine, where the local soil concentrations (Pb = 16300 mg/g d.w., Zn = 48800 mg/g d.w.) comply

with the Kelly Indices Guidelines definition of ―unusually heavy contamination‖ and exceed the

UK‘s Soil Guideline Values for domestic soils by orders of magnitude. We and others have

previously observed that chloragocytes not only have a phenomenal capacity to sequester and

partition metals into specific organelles, but must transport the metals to the organelles with

impressive efficiency whilst retaining sufficient functionality in, for example, the Pb-susceptible

haem biosynthesis pathway. Understanding how earthworms tolerate metals, and to better interpret

global transcriptome responses to metal exposures in terms of modes of action, would be promoted

by determining metal speciation in individual target cell types. But this goal presents a dilemma: how

to retain both the structural and compositional fidelities of the cells in close approximation to their in

vivo states? Cryo-preparative procedures offer analytical advantages but are less useful for critical

morphological examination of relatively large, complex, systems. We have exploited a promising

compromise technique involving gentle fixation of whole-earthworm pieces in 70% alcohol,

followed by methacrylate embedding and transverse sectioning in the range of 2 to 5 m? Sections

were mounted on films suspended across holes in aluminum planchettes, and imaged without staining

prior to metal mapping and micro-EXAFS. These high-resolution studies under a synchrotron source

of high coherence and brightness confirmed that chloragocytes (within the typhlosole and those

facing the coelomic cavity) are the main foci of Pb and Zn sequestration in earthworms. The metallo-

speciation findings were verified where possible with analyses of unfixed cryostat sections. The

methacrylate protocol produces ‗permanent preparations‘ offering the possibility of correlative

studies. Whilst synchrotron-based techniques will never achieve the status of being truly routine,

micro-focus techniques yield spatially resolved assessments of metal partitioning that compliment the

recent drive to fractionate cellular metal content in homogenised tissues. Acknowledgement: This

study was supported by a beamtime award (Expt. No. 4693) from Diamond Light Source Ltd.

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Effects of earthworm Dendrobaena veneta and low metals concentrations

on soil bacterial activity and functional diversity

Plytycz Barbara

1, Kuriaska Ligia

2, Klimek Malgorzata

1, Klimek Beata Anna

2, Kruk Jerzy

3,

Nikliska Maria2

1Institute of Zoology, Jagiellonian University, Krakow, Poland

2Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland;

3Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

It is well known that soil microbes and earthworms are main drivers of soil processes, but the

interaction between these two groups is still poorly understood. We have shown that Dendrobaena

veneta exposure to natural soil polluted with heavy metals led to disrupted balance between bacteria

inhabiting the earthworm coelomic cavity and the earthworm immune system based on coelomocytes

(amoebocytes and riboflavin-storing eleocytes), and that metal contamination affects also soil

bacterial flora. Therefore, it seems reasonable to continue studies on mutual interactions between

soil-inhabiting microbes and earthworms, which may be additionally affected by metal pollutions.

We also aimed to evaluate the influence of D. veneta in unpolluted or slightly metal polluted organic

soil on soil bacterial activity and the functional diversity. Soil samples from the commercial supplier

were soaked either with deionized water or with low concentrations of Ni, Zn or Pb ions (metal

concentrations of 2, 2 or 5 mg/kg, respectively). Adult D. veneta were introduced to half samples of

each metal treatment and kept unfed for 4 weeks. At the termination of experiments: 1) earthworm

coelomocytes and riboflavin content were analyzed by flow cytometry and spectrofluorometry; 2)

soil bacterial activity (average color development-AWCD), richness (R) and the functional diversity

(Shannon‘s index- H‘) were estimated using BIOLOG® Ecoplates; 3) metal concentrations were

measured in soils and in earthworm bodies. The results showed that Ni and Pb accumulated

significantly in worm bodies. Earthworm eleocytes and riboflavin content were affected by Ni and Pb

treatments. The bacterial activity (AWCD) was generally higher in soil temporarily inhabited with

worms than in soils without them. Presence of worms changed significantly microbial richness. The

functional diversity H‘ increased in soils with worms but the values did not differ significantly in

comparison with H‘ index for soils without worms. Metals have engendered different changes in the

bacterial parameters. The highest toxic effect was observed for lead treatment which significantly

reduced activity, richness and bacterial functional diversity. Different effects of worms and metals on

activity, richness and soil bacterial functional diversity need more comprehensive study of soil

macro-and microfauna interaction. Acknowledgements: Supported by No PB3502/PO1/32 and DS

759/INoS/2009.

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Bioreclamation of acidgoldmine tailings using Pontoscolex corethrurus

Prabha M.S. Swayam, Sharma Bhargavi, Balasubramanyam Priyanka,

Kale Radha D.

Mount Carmel College, (Autonomous), Bangalore University, Bangalore

Earthworms are considered to be bio-engineers. They are implied in pollution control, land

reclamation, bio-remediation, and as biomarkers of eco-toxicity. The studies have primarily

concentrated on the metal uptake and accumulation by Lumbricus, Eisenia and Pheretima species.

Pontoscolex corethrurus needs to be explored for restructuring the soil. This earthworm is a

voracious geophagous feeder and the physico-chemical properties of the soil ingested is transformed

considerably during its passage through gut. P.corethrurus also processes three pairs of calcium

glands which release or resorbed ions to/from the environment and change the pH of the medium and

make itself better adapted to the hostile soils. The present study enumerates the role of P .corethrurus

in bioremediation of the acidic gold mine tailings. Kolar gold mines were one of the three major gold

mining areas of India and gradually closed operations from 2001. The abandoned mine site now has

about 32 million tonnes of tailings dumps lying in the midst of township and occupying 10% of the

land area. The dust and leachate from dumps resulted in eco-toxicity and respiratory problems to the

ex-miners living in the vicinity. Hence, the present study was undertaken for restructuring of the

dump waste. The success of earthworm survival in the highly acidic tailings will enable restructuring

and converting the ‗spoil to soil‘ and support revegetation of the area. With this, the toxicity of the

area can be reduced and surface erosion and contamination of adjacent cultivation area prevented.

Mine tailings are highly acidic (pH 3.5-5.5) with zero organic matter. Pot culture with sandwich

layering of mine tailings with red soil and farmyard manure supported the survival of P.corethrurus

for the study period of 16 weeks. The earthworms ingested the minetailings as they burrowed through

changed from 5.5 to 7.5 and %OM from 0.031 to 1.86. Water holding capacity, available phosphate

and potassium increased and the bulk density decreased following P. corethrurus activity in the mine

soil. Thus, there was not only the mixing of layers through burrowing but also the restructuring while

passing through the earthworm gut. Thus, the physico-chemical characters of the toxic tailings were

brought closer to support plant viability. Increase in organic matter and water holding capacity is a

measure of improved soil fertility tailings but not lead. The metal concentration in the mine tailings

recorded a reduced concentration following earthworm activity. It can now be inferred that the

locally inhabiting P. corethrurus can effectively accumulate the metals and restructure the gold mine

tailings for reclamation and revegetation and thus alleviate ecotoxicity and health hazards.

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Heavy metal (Cd, Cu, Pb, Zn and Mn) bioaccumulation by the

earthworms (Eudrilus eugeniae, Eisenia fetida and Perionyx excavatus)

during vermicomposting of urban waste

Reddy Vikram M., Pattnaik Swati

Department of Ecology and Environmental Sciences, Pondicherry University, Pondicherry 605 014, India

The present study revealed bioaccumulation of Cd, Pb, Zn, Cu and Mn by the mature (clitellate)

specimens of three earthworm species - Eudrilus eugeniae, Eisenia fetida and Perionyx excavatus

during the vermicomposting processes of three different urban wastes – Municipal Solid Waste

(MSW), Vegetable Market Waste(VMW) and Flower Waste (FW), carried out for sixty days. The

metals of earthworm tissue and the substrates were analyzed at every 15 days interval. The metal

concentrations in earthworm tissue and its bioaccumulation factor (BAF) were significantly higher in

E. eugeniae than that in E. fetida and P. excavatus cultured in the three substrates seperately

(P<0.05); and were low at the beginning i.e., at 15 days of the vermicomposting process and

increased gradually towards the end of the procees in the ranking order of 15 days<30 days<45

days<60 days. The concentrtions of Cd and Cu were higher in the earthworms cultured in MSW and

that of Pb, Zn and Mn were higher in those cultured in VMW. The BAF of the metals followed the

ranking order of Cd>Zn>Pb>Cu>Mn in the earthworm species and the BAF of the metals except that

of Cd were less than one in the tissue of three earthworm species, across the substrates and the time

intervals; while, it was more than one in case of Cd.

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Metals in earthworm casts are more mobile but earthworm mucus reduces

mobility

Sizmur Tom

1, Palumbo-Roe Barbara

2, Hodson Mark E.

1

1University of Reading

2British Geological Survey

Human activities have resulted in an increase in the concentrations of metals in both urban and rural

soils which can lead to toxic effects on flora and fauna. Generally earthworms appear to increase the

mobility and availability of metals but the mechanisms for this are unclear. Earthworms burrow in

the soil and create casts that are chemically, biologically and physically different to the surrounding

soil. In addition, they also release mucus into the soil solution which has been shown to increase the

uptake of metals into plants when added to hydroponic solutions (Zhang et al. Env. Poll. 157, 2009).

In order to explain the mechanisms behind the impacts of earthworms on metal mobility in soils it is

necessary to separate the effects that are due to passage through the gut from the effects due to the

excretion of mucus. We used Lumbricus terrestris (L.) and soil that was amended with Cu (120

mg/kg), Pb (988 mg/kg) and Zn (241 mg/kg) fifteen years ago. One experiment aimed at determining

the effect of earthworm mucus on metal mobility compared metal extractability from soil using a

dilute solution of earthworm mucus and a control solution of deionised water. A second experiment

compared water soluble metals in earthworm casts with bulk earthworm inhabited soil and

earthworm-free control soil. Passage of soil through the earthworm gut significantly increased the

concentrations of water soluble Cu (from 0.8 to 2.3 mg/kg), Pb (from 1.7 to 3.7 mg/kg) and Zn (from

1.3 to 2.9) by a factor of 2.9, 2.2 and 2.1 respectively. However, significantly less Cu (from 0.26 to

0.25 mg/kg) and Pb (from 0.51 to 0.34 mg/kg) were extractable with the mucus solution than with

deionised water, although this effect is small compared to that of the gut. The greater mobility of

metals in the casts is probably due to the release of organically bound metals during organic matter

degradation in the gut. It is not clear what process is responsible for the reduced extractability of

metals by the mucus solution. Before one can comment on the environmental significance of these

findings, more information is required concerning the longevity of each of these effects and how

diluted they become in the soil environment.

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Earthworm survival in oil contaminated soil

Trujillo Capistrán Juan F.

1, Zavala Cruz Joel

2, Guerrero-Peña Armando

2,

Ortiz Ceballos Angel I.1

1Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana. Xalapa, Veracruz, Mexico

2Colegio de Posgraduados Campus Tabasco. H. Cárdenas, Tabasco, Mexico

In the southwest of Mexico the contamination of soil by hydrocarbons due to the extraction

conduction and transport of crude petroleum is frequent. In diverse studies on environmental risks,

the earthworms have been used like toxicity indicators. The earthworms are associated with the

health of soil and its absence can be an indicator of degradation of the soils. The knowledge about the

sensitivity of earthworms to the contamination of soil by crude petroleum and its ability to survive in

bioremediated soil is limited. The work objective was to evaluate the populations of earthworms in a

contaminated soil (Arenosols) with crude petroleum. In the low basin of the Tonala-Tabasco River a

contaminated arenosols soil was selected; it was because of a crude petroleum spill. In a

contamination gradient, from the spill, 15 monoliths were realized in the three transects (50 m of

length and 5 m of separation). In each, 5 monoliths became (25x25x40 cm). The monoliths separated

in four layers (10 cm) and in each the earthworms were collected manually (diversity, distribution,

abundance and biomass) and samples were taken from soil to determine the physical and chemical

properties and the total hydrocarbon content. The Pontoscolex corethrurus earthworm was the

dominant species. The distribution of the earthworms to the contamination gradient was similar with

an abundance and biomass average of 296.5 worms/m2 and 55.9 g/m

2, respectively. The vertical

distribution (profile) varied with the contamination gradient. The preliminary conclusion suggests a

tolerance of the earthworms to inhabit contaminated soil with intemperate crude petroleum exceeding

the official norm.

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Increasing temperature enhances Cd toxicity and

accumulation in E. Andrei

Voua Otomo P., Reinecke SA, Reinecke AJ

Stellenbosch University

To determine whether the toxicity of cadmium to earthworms is affected by temperature, adult

specimens of Eisenia andrei were exposed in OECD soil to a series of Cd concentrations (0, 250,

500, 750, 1000 g/kg) and incubated for 28 days in three different temperatures (15, 20 and 25 oC).

During the exposure period, earthworms‘ biomass, reproduction and survival were assessed weekly.

Results indicated that, at each temperature, the biomass decreased as metal concentration increased.

However, higher temperature led to higher biomass loss. The highest biomass loss was observed at

1000 mg/kg Cd for all temperatures. These losses were 42.12% at 15 oC; 50.98% at 20

oC and

87.72% at 25 oC (means for all replicates). Survival data also indicated that temperature enhanced

cadmium toxicity. At 15oC, mortality (3.3%) only occurred at 1000 mg/kg Cd. At 20

oC mortality was

recorded at 1000 (3.3%) and 750 mg/kg Cd (6.6%). At 25oC, mortality occurred at 1000 (66.6%),

750 (56.6%) and 500 mg/kg Cd (13.3%). An LC50 of 725.46 mg/kg, for E. andrei exposed to Cd

could only be determined at 25 °C because the mortality rates in the other temperatures were too low.

Reproduction was completely inhibited in all Cd exposures. The incubation period of cocoons (that

were only recorded in the control treatments) decreased as temperature increased (84 days at 15 oC,

21 days at 20oC, and 13 days at 25

oC). Inversely, the mean number of hatchlings per cocoon

increased as temperature increased (0.5 at 15 oC, 1.56 at 20

oC, and 1.79 at 25

oC). The earthworms

were analyzed to determine their Cd body contents at the end of the exposure period. These results

revealed a dose response accumulation pattern and also an increase of Cd body burden in earthworms

as temperature increased from 15 oC to 20

oC and to 25

oC. It is concluded that increasing

temperature enhances Cd toxicity and accumulation in E. andrei.

264


5th to 10



Impacts of earthworms (Eisenia fetida and Amynthas morrisi) and organic

matter on the availabilities of Zn, Cu, Cd and Pb in contaminated soils by

heavy metals in Southern China

Zhang Chi

1, Chen Xu Fei

1, Li Huan-Huan

1, Lavelle Patrick

2, Dai Jun

1

1College of Natural Resources and Environment, South China Agricultural University, 510642

2UMR Bioemco, IRD, 32 rue Henri Varagnat, Bondy Cedex 93143, France

Laboratory cultures experiments were conducted to evaluate the impacts of earthworm and organic

matter on the availabilities of Zn, Cu, Cd and Pb with multiple heavy metals contaminated soils in

Southern China for 60 days. Two species of earthworms, Eisenia fetida, and a native earthworm,

Amynthas morrisi were introduced into the contaminated soil, which were taken from Dabaoshan

mine in Southern China. And the total metals contents of the soil are 394 mg·kg-1

, 405 mg·kg-1

, 439

mg·kg-1

, and 0.64 mg·kg-1

for Cu, Zn, Pb, and Cd respectively. All the following treatments

combinations were implemented in six treatments: (1) 100% soil, (2) 100% soil + Eisenia fetida, (3)

100% soil+ Amynthas morrisi, (4)10% organic matter (mixture of coco bran and cattle dung) + 90%

soil, (5) 10% organic matter +90% soil+ Eisenia fetida, (6) 10% organic matter + 90%soil +

Amynthas morrisi. Both of surface and internal casts of two species earthworm were separated from

soil and collected respectively. pH, total contents of Zn, Cu, Cd, Pb and their DTPA-extractable

fractions were measured respectively. Results showed that soil pH ranged from 4.71 to 6.50 and they

increased significantly in 100% soil treatment with earthworm at the end of the culture (p<0.05),

however, no variation has been observed in 10% OM treatments. For total heavy metal, cast of E.

fetida have higher Cu and Pb compared to A. morrisi, while lower Zn and Cd. For DTPA-extractable

heavy metal, results showed DTPA-Cd in cast of A. morrisi >E. fetida > control soil (p<0.05), while

DTPA-Zn, Cu, Pb in cast of control > or ≈E. fetida > or ≈ A. morrisi. As conclusion, our study

confirmed different effects of two epigeic earthworm species on total heavy metal contents and their

availabilities in contaminated soil with/without organic matter and proved A. morrisi could be a more

superior to be used in the bio-remediation of heavy metal contaminated soil.

265


5th to 10


Session 8

Vermicomposting and Waste Management

266


5th to 10



Effects of Vermicomposts and Aqueous Extracts from Vermicomposts

('Teas') on Plant Diseases and Pests

Edwards Clive A.1, Arancon Norman2

1 The Ohio State University, USA,

2 University of Hawaii-Hilo, USA.

Vermicomposts are produced by interactions between earthworms and microorganisms, in breaking

down food and animal organic wastes, to produce finely-divided, fully-stabilized soil amendments or

plant growth media, containing available plant nutrients and plant growth regulators. They have

dramatic effects on crop germination, growth, flowering and fruiting. Vermicompost aqueous

extracts (‗teas‘) were produced in commercial brewing equipment, from 20 parts vermicompost to 80

parts water (v.v.), to produce a 20% extract, that was diluted to 10% and 5% and compared with a

water control. Substituting vermicomposts into equal amounts of a plant growth medium (MetroMix

360) decreased attacks by the root pathogens Pythium ultimum, Rhizoctonia solani, Verticillium sp.,

and powdery mildew almost completely, in laboratory and field experiments. Applying soil drenches

of vermicompost aqueous extracts(‗teas‘) to plants, suppressed attacks by the root pathogens,

Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani and Pythium ultimum, on tomatoes

and cucumbers very dramatically. Leaf sprays with ‗teas‘ decreased attacks by foliar pathogens, such

as Plectosporium tabacinum, Botrytis cinerarea, Sclerotinia rolfsii and Verticillium sp., on tomatoes

and cucumbers almost completely, in greenhouse experiments. The pathogen suppression must have

been microbially-mediated, because sterilization removed the suppressive activity. The suppression

mechanism could have been due to: microbial competition, antagonism, anti-fungal activity or

antibiotics produced by bacteria. Substitution of 10%, 20% or 40% vermicomposts into the plant

growth media (MetroMix 360), suppressed attacks by arthropod pests such as aphids, two-spotted

spider mites, mealy bugs and cabbage white caterpillars, caged over cucumbers and tomatoes, almost

completely. Vermicompost aqueous extracts (‗teas‘) applied as to the plants as soil drenches

suppressed numbers of aphids, mealy bugs, two-spotted spider mites, cabbage white caterpillars,

tobacco hornworms and cucumber beetles, caged over the plants, almost completely in greenhouse

tests. The degree of suppression was correlated with the ‗tea‘ concentration. The most likely

mechanism for the arthropod suppression appeared to be the uptake of water-soluble phenols from

vermicomposts and 'teas' into the plant tissues, making them very unattractive to the pests, decreasing

pest reproduction and killing pests.

267


5th to 10


Oral – Session 8

Comparison of co-composting with and without Eisenia fetida for

bioremediation of distillery sludge

Kaur Arvinder, Singh Jaswinder, Viga Adarsh Pal, Rup Pushpinder Jai

Guru Nanak Dev University, India.

The aim of the present study was bioremediation of toxic distillery sludge into a soil enriching

material. The sludge produced from the spent wash of distilleries has a high BOD, COD and nutrient

imbalance and depresses soil flora and fauna of the disposal sites and pollutes environment for a long

duration. This sludge was mixed with a complementary waste the cattle dung and subjected to co-

composting with (V) and without (T) Eisenia fetida. The sludge was mixed with cattle dung in the

ratio of 0:100 (V1, T1), 12.5: 87.5 (V2, T2), 25: 75 (V3, T3), 50:50 (V4, T4), 75:25 (V5, T5) and

100:0 (V6, T6) w/w on dry weight basis and efficiency of vermicomposting (with E. fetida) and

aerobic composting (without E. fetida) was adjudged with respect to time taken for biodegradation

and quality of the end products from the two technologies. The distillery sludge was observed to be

very toxic to earthworms even after precomposting of three weeks but the proportion of cattle dung

in the mixture enhanced its acceptability for E. fetida. Survival rate, growth rate, onset of maturity,

cocoon production and population buildup decreased with decreasing concentration of cattle dung in

the mixture. Minimum mortality and maximum population buildup were observed in the 12.5: 87.5

mixture of the sludge and cattle dung. On the basis of response surface design the concentration

giving highest number of worms, cocoons and hatchlings came out to be 21.11%, 24.51% and

17.19% respectively. The weight of cocoons as well as hatchlings also showed a negative correlation

(r = -0.78) with the amount of distillery sludge in the mixture. Organic carbon declined significantly

(p<0.05) in the vermicompost as compared to a small decline in the products of traditional aerobic

composting. Nitrogen, phosphorous, sodium and pH increased in the products of vermicomposting

but decreased in the products of traditional aerobic composting. However, electrical conductivity and

potassium showed opposite trends in the products of these two techniques. An increase was observed

in the contents of transition metals in the products of both the techniques but these were with in the

international limits of heavy metals in the manures. The comparison clearly indicated that

vermicomposting was a better technology over traditional aerobic composting for bioremediation of

toxic distillery sludge as it gave a better product in five months time.

268


5th to 10


Oral – Session 8

Extraction condition influenced vermicompost tea function

Dai Guohua, Li Jing, Sun Zhenjun

China Agriculture University, China.

In recent years, "vermicompost tea" was proved to have the function to accelerate crop nutrient

absorption, decompose harmful toxins, and restrain soil-borne pathogen microbes. What components

in vermicompost tea to bring these functions? Microorganisms, special nutrients or comprehensive

substances? We try to give answer in this paper. The results showed vermicompost tea made by raw

material from cow manure contained higher necessary nutrients and trace element and easily

absorbed by plant, for example, soluble P, NO3 - N and exchangeable Mg, K, Ca,, etc. In addition,

soluble salt content, cationic exchange properties and humus acid content were significantly

increased in vermicompost tea. By the joint action of microbes, organic matter converted into the

stable humus complex. Some soluble humus acid can not only affect plant nutrition absorption, and

can affect protein synthesis. In the microflora vermicompost was rich in bacteria, fungi and

actinomycetes. These microbes plays a very important role, not only make the complex material

mineralized into effective nutrients easily absorbed by plants, but also some produced bioactive

substances, such as sugar, amino acid, vitamin etc in it. Microbial flora composition and content of

nutrient levels have significant influence with different extraction temperature and extraction time

from fresh vermicompost. On the conditions of 25 °C extraction temperature and 36h extraction time,

the solution has the highest content of microorganisms and then the microbe quantity fell down

rapidly. While the higher extraction temperature, bacteria and fungi vermicompost tea had significant

adverse effects.

269


5th to 10


Oral – Session 8

Vermicomposting of sewage sludge: Effect of PAM (Polyacrylamide) on

the growth and reproduction of the earthworm Eisenia foetida

Guo Feifan, Zhang Qin, Sun Zhenjun


Polyacrylamide (PAM) is a general designation of homopolymer and multipolymer of Acrylamide

(AM). Owing to own characteristic of molecular constitution, PAM is apt to produce modifier,

through the charge neutralization and inter-particle bridging action, So It has been extensively used

to in every field and was extolled as multipurpose product. Because of good flocculation capability of

PAM, it is still the water treatment agent which was used much in water treatment filed at home and

abroad, especially for wastewater treatment. The total usage of PAM was 65×104 t all over the world

in 2005, and 40 percent of it has been used in water treatment field. Research indicates that the

concentration of PAM in sewage sludge can reach 1000mg/kg (dry weight). In the past the PAM was

considered as stable, safe and nontoxic substance. In fact, the PAM will produce various oligomer

and monomer, under influence of physical, chemical and biologic factor in nature environment, and

AM monomers have been shown clinically to act as animal carcinogens and neurotoxins. Therefore

this study addressed the possible role of PAM to earthworm during vermicomposting through acute

and subacute toxicity testing. The result shows the PAM and AM have effect on not only the living

and growth but also reproduction of the earthworm when they reached a certain concentration.

270


5th to 10


Oral – Session 8

Vermitechnology for fast conversion of toxic wastes of a soft drink

industry into quality manure

Singh Jaswinder1, Kaur Arvinder

2

1 Department of Zoology, Khalsa College, Amritsar, Punjab, India,

2 Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, India.

Large quantities of highly toxic spent carbon and chemical sludge are generated during the

production of soft drinks. The industries generally dump them in or out of the premises in low lying

areas. An attempt has been made to convert these wastes into nutrient rich manures through

vermicomposting with Eisenia fetida. Both spent carbon and chemical sludge were very toxic to

earthworms so these were mixed with cattle dung in various proportions. But 100% mortality of

worms was observed even in 20:80 % mixture of spent carbon with cattle dung and 50:50 % mixture

of chemical sludge with cattle dung. Therefore mixtures with higher percentage of each waste were

discarded. Two mixtures per 2 Kg of each waste i.e 5% and 10% for spent carbon and 20% and 40%

for chemical sludge were selected and inoculated with 20 worms. These mixtures were degraded

after a long time of 6 months so another experiment was run with a variation in weight of earthworms

(g) per Kg waste for reducing the time of recycling the wastes. The worms were inoculated at 7.5,

12.5 and 25 g/ Kg waste. It was noticed that recycling time and decline in organic carbon showed a

positive correlation (r = 0.98) with weight of worms/ Kg in both the wastes. Mixture with 5% spent

carbon and 20% chemical sludge were degraded respectively after 70 and 95 days with 25 g worms/

Kg mixture. However, chemical analysis showed that in final products nitrogen, phosphorous and

sodium content was maximum in 12.5 g worms/ Kg feed mixture. Maximum increase in electrical

conductivity was observed in 12.5 g worms/ Kg spent carbon mixture but in chemical sludge

maximum increase was in 7.5 g worms / Kg mixture. pH declined significantly from the initial feed

mixture ( p < 0.05) and maximum decrease was in 25 g worms/ Kg mixtures. Although potassium

and transition metals showed a decline over initial in all the products but the decline showed a

positive correlation with the weight of worms in the mixtures.

271


5th to 10


Oral – Session 8

Mechanisms of reduction of total coliform numbers during

vermicomposting

Monroy Fernando, Aira Manuel, Domínguez Jorge

Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Spain.

During vermicomposting of organic waste, the interactions between epigeic earthworms and the

detrital microbial community lead to decreases in the abundance of some potentially pathogenic

microorganisms. Despite its importance, little is known about the mechanisms involved and the

factors that affect the intensity of this effect. We carried out three experiments to test the effect of the

earthworm Eisenia fetida on total coliform numbers in pig slurry. We firstly applied low and high

doses of pig slurry to small scale vermireactors with and without earthworms. We found that E. fetida

significantly reduced total coliform numbers after two weeks, but only in the low dose vermireactors.

In a subsequent feeding experiment in mesocosms, we observed that the coliform population was

reduced by 98% after passage through the earthworms‘ guts, which suggests that digestive processes

in the gut of E. fetida are the main factors involved in the decrease in total coliforms observed in the

low dose vermireactors. Decreases in total coliform numbers were not related to decreases in

bacterial biomass, which indicates a specific negative effect of earthworms on the coliforms. In the

third experiment, we tested the indirect effect of earthworms on total coliforms by inoculating pig

slurry with either 2 or 10% vermicompost. The addition of vermicompost did not affect the number

of coliforms either after 15, 30 or 60 days, which supports the idea that this bacterial group is more

affected by the passage through the gut of E. fetida than by interactions with the earthworm-shaped

microbial community.

272


5th to 10


Oral – Session 8

Efficacy of vermiwash-smeared mulberry leaves on cocoon characters of

multivoltine hybrid mulberry silkworm Bombyx mori L.: Kolar Gold

(K.G.) Race

Rawgol Yatheesh K. 1, Maddi Priyadarshini P.

1, Sharma Venugopal

2, Kale Radha D.

3

1 Christ University, Bangalore, India,

2 SAS Analyst, Bangalore, India,

3 Mount Carmel College, Bangalore, India.

The effect of vermiwash and vermicompost in augmenting the growth process in a variety of plants is

amply documented. Biological role of feed supplements like semi-synthetic diets and mulberry

grown through vermicompost administration on the growth and cocoon characters of silkworms is

also equally well known. In the present investigation, the effect of fresh and two week old vermiwash

of two epigeic earthworms Eudrilus eugeniae and Perionyx sp. on the cocoon characters of the

mulberry silkworm, multivoltine hybrid Kolar Gold race was studied. Mulberry leaves were collected

from a field where different treatments were given to the existing three year old mulberry crop. The

treatments to mulberry plants included spot application of 1 and 2kg. vermicompost, 30g NPK

(300N:120P:120K) and Farm yard Manure (1kg) respectively. One set of the plants were sprayed

with vermicompost extract (1kg. fresh vermicompost suspended in ten litres of fresh water and left

for 72hr. before decanting to use as spray), one more set of plants received vermicompost brew (1kg.

Vermicompost was treated with water at 100°C and slowly filtered through a gauge filter to get the

brew to use as spray to plants) and finally spraying of water to third set served as control. The leaves

collected from these treatments were used as feed to different batches of silkworms of fourth instar

immediately after the third molt. The silkworms were divided into two groups and one was fed with

the leaves collected from the plants exposed to different treatments smeared with vermiwash [1kg.

earthworms were immersed in one litre warm water for 30 sec. in a tray and later transferred to

another tray containing water at room temperature (26 to 28°C) before releasing them back into

bins.The fluid in both the trays were mixed and this served as vermiwash] and the other group was

fed on the leaves without smearing with vermiwash (untreated leaves). The parameters recorded

included weights of larvae, silkglands, cocoons, pupae, shell and floss. Vermiwash- smeared

mulberry leaves grown with vermicompost and sprayed with brew showed significant influence on

the cocoon characters when compared to other treatments. The data is analysed using SAS program

to show the levels of significance.

273


5th to 10


Oral – Session 8

Earthworm effect on gaseous emissions during vermifiltration of animal

wastewater

Saja Luth1, Robin Paul

2, Germain Philippe

2, Lecomte Marcel

2, Landrain Brigitte

3, Cluzeau

Daniel 4

1 Université de Rennes 1, France & INRA de Rennes, France,

2 INRA de Rennes, Agrocampus-Ouest, France,

3 Station Expérimentale de Guernevez, Chambres d‘Agriculture de Bretagne, France,

4 Université de Rennes 1, France.

Vermifiltration is used to treat wastewater. As wastewater of animal farms is rich in available

nitrogen and carbon, treatment of wastewater can be a source of ammonia (NH3), nitrous oxide (N2O)

and methane (CH4). Earthworms have the advantage of mixing and transforming nitrogen and carbon

without consuming additional energy. However this technology should not increase gaseous

emissions. The objective of this paper is to analyze the specific role of earthworms on emissions of

NH3, N2O and CO2 during vermifiltration of animal wastewater. The experiment used fresh slurry of

swine wastewater and the material of a vermifilter running with woodchips. Three levels of slurry

input were added on eighteen mesocosms, with or without earthworms, during one month. The

earthworm abundance was measured three times, and the gas emissions five times, with the closed

chamber method. The results showed that earthworms decrease the NH3 and N2O emission when

slurry levels are normal or high. A small increase of N2O emission was observed with the

earthworms associated to the lowest input of slurry. These effects are attributed to the role of

earthworms in mixing the fresh organic matter after input and maintaining a connected free air space

inside the porous media. We suggest that earthworms abundance could be used as a bioindicator of

low energy and low green house gas emissions when slurry systems are converted to fresh slurry with

water recycling.

274


5th to 10


Oral – Session 8

Vermicomposting in Businesses and Institutions in N. America

Sherman Rhonda

North Caroline State University, USA.

Vermicomposting converts organic waste materials, otherwise destined for landfills or incineration,

into a valuable soil amendment called vermicompost that promotes plant growth and prevents plant

diseases and pests. Thus, vermicomposting has environmental and economic potential. The primary

organic components of waste streams for businesses and institutions are food residuals, paper, and

yard debris. Some agencies already have their paper and yard waste collected for recycling (by their

municipality or local businesses) and they want to focus on food waste recycling. Others desire to

recycle all three streams on site. Businesses and institutions are becoming environmentally-conscious

and looking for ways to cut costs. Vermicomposting is a viable method of diverting the organic

portion of their waste streams away from disposal and instead converting it to a value-added product.

More and more businesses and institutions are vermicomposting to manage their organic waste.

Vermicomposting helps them save solid waste collection and disposal fees, and wastewater

processing costs. Organic materials are vermicomposted on-site or transported to a centralized

facility. Medium-scale vermicomposting takes place at businesses and institutions, such as prisons,

universities, hospitals, office buildings, schools, and military bases. Vermicomposting provides a

cost-effective and environmentally beneficial solution for managing food waste, paper products, and

yard debris on-site. Vermicomposting helps businesses and institutions save solid waste collection

and disposal fees, water usage costs, and wastewater disposal expenses. Brief case studies of a

variety of businesses and industries that are vermicomposting food and paper waste will be

presented.

275


5th to 10


Oral – Session 8

Vermicomposting of biosolids: earthworm population growth and

agronomic characteristics

Solis Mejía Ludibeth, Islas Espinoza Marina, Esteller Alberich Maria Vicenta

Universidad Autónoma del Estado de Mexico, Mexico.

The biosolids that came from the wastewater treatment are a severe source of environmental

contamination due the lack of an adequate disposal management. The vermicomposting can be used

to enrich and mineralize biosolids in order to amend agricultural soils. The aim of this study was to

estimate the adaptation of the earthworm Eisenia foetida to different mixtures of biosolids with cow

manure and evaluate the quality of the vermicompost as fertilizer. Five treatments with three

replicates were prepared in plastic containers (3 L) perforated in the base. The treatments contained

biosolids and cow manure in a proportion of 70:30 (A), 80:20 (B), 90:10 (C), 95:5 (D), and 100:0

(E), with a moisture content of 60-70%, and were incubated at an average temperature of 15 ° C. The

dried mass was 0.8 kg per container, with 50 mature earthworms each. Following 50 days of

composting, cocoons, juveniles and mature earthworms were counted. Vermicompost was sampled

and pH, bulk density, organic matter, organic C, total N, inorganic N and available P and C/N were

determined. E. foetida could not tolerate the treatments D and E, likely due to ammonification

processes and lack of oxygen of the biosolids. The treatment B had the higher survival (100%) and

reproduction (77 mature, 18 juveniles and 150 cocoons). In the treatment C were detected the highest

amounts of N and P available (1950.89 and 2203.96 mg kg-1, respectively) and a C/N mineralized

product of 12.46, making it the better product as fertilizer; the values of bulk density (0.64 g cm-3),

organic matter (54.83%), organic C (31.80%) and total N (2.55%) were within the ranges established

for a vermicompost. With the exception of P (1882.15, 1929.18 y 2310.63 mg kg-1 in A, B and C,

respectively), the differences between the physical and chemical parameters of the treatments were

statistically significant (p<0.05), which indicated that different proportions of biosolids and cow

manure had different vermicompost characteristics. Therefore, the vermicomposting of biosolids and

cow manure with E. foetida can produce amendments to improve soil fertility and nutrient

availability in agricultural soils.

276


5th to 10


Oral – Session 8

Suppressive compost: Vermicompost enriched with biocontrol agents

Thavamani Uma1, Bagyaraj D.J.

2, Kale Radha D.

1

1 Dept. of Biotechnology, Mount Carmel College, Bangalore, Karnataka , India,

2 Centre for Natural Biological Resources and Community Development, Bangalore, Karnataka, India.

Soil-borne diseases result from a reduction of biodiversity of soil organisms. Restoring beneficial organisms

that attack, repel or otherwise antagonize disease causing pathogen will render soil disease suppression. Plants

growing in disease- suppressive soil resist diseases much better than in soils low in biological diversity. The

introduction of a food base with the biocontrol agent that supports its activity without stimulating the activity of

pathogens can be a recent approach to govern rhizosphere competency. The application of organic wastes

directly to soil leads to many problems such as immobilization of nutrients, phytotoxicity etc. Compost quality

determines its effectiveness at suppressing soil-borne plant diseases. Vermicomposting is the use of

earthworms for composting of organic residues. The information on vermicompost enriched with biocontrol

microorganisms (suppressive vermicompost) is meager. The analysis of how microbial consortia in complex or

compost amended substrates interact with one another, the pathogen, or the host to facilitate disease

suppression has always been a challenge. In the present study following three objectives was investigated: a)

the shelf life of biocontrol agents in vermicompost maintained at three different moisture levels. b) Influence of

gauge size of polybags on the colony forming units of bio- inoculants in vermicompost and c) to compare

sterilized vs. unsterilized vermicompost as the carrier for biocontrol agents. The biocontrol agents used were

Pseudomonas fluorescens, Streptomyces sp. and Trichoderma viride; they were multiplied on King‘s B, Ken

Knight‘s and Trichoderma specific medium respectively. The vermicompost was taken in 100g lots in

polythene covers, sealed and thoroughly mixed . Three to four pin holes were made with sterile needle and

incubated at room temperature. Initial population and the population at fortnightly intervals up to fifteen

months were determined by serial dilution pour plate method. To determine the favourable moisture level for

suppressive compost Phosphate buffer was added to obtain three different moisture levels viz. 15, 20, and 25

%. The results showed that the biocontrol agents at 20 and 15% moisture levels increased up to 30 days of

incubation, thereafter it declined. The cfu/g of the all the biocontrol agents was significantly higher at 25%

moisture level at all intervals of sampling. Thus it was concluded that 25% moisture level is more suited for

longer shelf life of all the three biocontrol agents under study. To study the influence of gauge size of polybags

on the vermicompost enriched with biocontrol agents the moisture content of vermicompost adjusted to 25%

was taken in 100g lots in polythene covers of three different gauge size viz., 39, 47 and 50. The result obtained

showed that the biocontrol agents had a longer shelf life with higher cfu/g in the thickest polybags i.e., 39

gauge. Thus it was concluded that 39 gauge polybag is better suited for longer shelf life of the suppressive

vermicompost. Finally sterilized vs. unsterilized vermicompost were compared for the ability to harbor

biocontrol agents using 39 gauge polybags and vermicompost at 25 % moisture level. The initial population of

Pseudomonas fluorescens, Streptomyces sp. and Trichoderma viride were 1.42 x 108, 6.00 x 10

6, and 16.75 x

105 cfu/g respectively. According to the results obtained using sterilized and unsterilized vermicompost, the

colony forming units of biocontrol agents were significantly higher in case of sterilized vermicompost as

compared to unsterilized vermicompost with respect to all the three biocontrol agents. It can be concluded that

25% moisture level, sterilized vermicompost packed in 39 gauge polybags is best suited for the longer shelf life

of suppressive vermicompost.

277


5th to 10



Opening the black box of vermicomposting: relationships of earthworms

and microorganisms

Aira Manuel, Gómez Brandón Maria, Domínguez Jorge

Universidad de Vigo, Spain.

Microorganisms are the main decomposers, but earthworms also affect decomposition directly by

feeding on and digesting organic matter, or indirectly through their interactions with the microflora,

by stimulation or depression of the microflora biomass and activity. Here we present the results of

three experiments which tried to separate between the direct and indirect effects of earthworm on

decomposition of organic matter. We used the vermicomposting process as a model to study the

relationships of earthworms and microorganisms. The first experiment studied the whole process of

vermicomposting, using Eisenia fetida and pig manure in continuous feeding vermireactors; these

vermireactors were made of independent layers which allowed us to date them. Earthworms

promoted significant increases in both microbial biomass and activity when they were present in the

layers, and after that, in aged layers where earthworm were absent, microbial stabilization (i.e. low

microbial biomass and activity) occurred. Further, community physiological profiles (Biolog

Ecoplate) revealed that earthworms promoted significant alterations in the physiological capabilities

of microorganisms. As consequence, carbon mineralization was significant enhanced. In a second

experiment, we inoculated pig manure with two doses of vermicomposts (2.5 and 10%) obtained

from laboratory cultures of E. fetida, E. andrei and Eudrilus eugeniae. By doing this, we were able to

test the indirect effect of earthworms, that is, whether an earthworm processed substrate can modify

organic matter decomposition or whether it is necessary the earthworm presence to achieve the same

results of the first experiment. We found that inoculation of vermicompost produced the same

increases in microbial biomass and activity, and although these increments were not as higher as in

presence of earthworms, we can state that indirect effects of earthworm are strong enough to alter the

dynamics of organic matter decomposition. In the third experiment, we analyzed the gut content of

four earthworm species (E. fetida, E. andrei, E. eugeniae and Octodrilus complanatus) which were

fed with pig slurry. We determined the number of bacterial cells by fluorescence microscopy to

understand the direct effect of earthworms on microorganisms. We found that only O. complanatus

reduced bacterial counts, whereas there were no differences between pig manure and the gut content

of the other earthworm species.

278


5th to 10



Enzymatic activity decreases associated with the fall of microbial biomass

during aging of vermicomposting



Vermicomposting is the biooxidation and stabilization of organic matter involving the joint action of

earthworms and microorganisms, thereby turning wastes into a valuable soil amendment called

vermicompost. Studies have focused on the changes in the type of substrates available before and

after vermicomposting, but little is known on how these changes take place, especially those changes

related with maturation of vermicompost. In the present study, the effects of vermicompost aging

especially patterns of changes in microbial biomass and enzyme activities were investigated because

these parameters can control the quality of the resulting vermicompost. To do this we analyzed the

pig slurry once earthworms have initially processed it (fresh vermicompost). We incubated 16 week-

old vermicompost (fresh vermicompost) and sampled it after 15, 30, 45 and 60 days analyzing

microbial biomass and activity (assessed as microbial biomass-N and basal respiration respectively),

and four enzymatic activities (-glucosidase, cellulase, protease, alkaline phosphatase). Aging of

vermicompost resulted in decreases of microbial biomass and activity. Three of the four enzymes

analyzed also showed decrease. An initial increase followed by a rapid decrease in alkaline

phosphatase was also recorded. High and significant correlations between microbial biomass and -

glucosidase (r = 0.62, P<0.001), cellulase (r = 0.56, P<0.01) and protease (r = 0.82, P<0.001) were

found. Results suggest that there may be two steps involved in the aging dynamics of vermicompost

with regards to extracellular enzyme activity; the first step was characterized by a decrease in

microbial populations, which resulted in a reduction in the synthesis of new enzymes. The second

step was the degradation of the pool of remaining enzymes. This dynamic does not seem to be

affected by earthworms because similar decaying patterns of microbial biomass and activity were

found in substrate where earthworms were present.

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Earthworms enhanced cellulose decomposition during vermicompost by

promoting of fungal biomass

Aira Manuel, Lazcano Cristina, Domínguez Jorge


Cellulose is the most abundant polymer in nature and constitutes a large pool of carbon for

microorganisms, the main agents responsible for organic matter decomposition. Cellulolysis occurs

as the result of the combined action of fungi and bacteria with different requirements. Earthworms

influence decomposition indirectly by affecting microbial population structure and dynamics, and

also directly because the guts of some species possess cellulolytic activity. Here we assess whether

the earthworm Eisenia fetida (Savigny 1826) digests cellulose directly (i.e. with its associated gut

microflora), and also whether the effects of E. fetida on microbial biomass and activity lead to a

change in the equilibrium between fungi and bacteria. By enhancing fungal communities E. fetida

would presumably trigger more efficient cellulose decomposition. To evaluate the role of E. fetida in

cellulose decomposition we carried out an experiment in which pig slurry, a microbial-rich substrate,

was treated in small-scale vermireactors with and without earthworms. Five samples of substrate per

module were taken at random and gently mixed for biochemical analyses, i.e. i.e. cellulose and

hemicellulose content, microbial biomass C (Cmic), ergosterol content, and β-glucosidase and

cellulose activities. The presence of earthworms in vermireactors significantly increased the rate of

cellulose decomposition (0.43% and 0.26% cellulose loss day-1, with and without earthworms

respectively). However the direct contribution of E. fetida to degradation of cellulose was not

significant, although its presence increased microbial biomass (Cmic) and enzyme activity (cellulase

and β-glucosidase). Surprisingly, as fungi may be part of the diet of earthworms, the activity of E.

fetida triggered fungal growth during vermicomposting. We suggest that this activation is a key step

leading to more intense and efficient cellulolysis during vermicomposting of organic wastes.

280


5th to 10



Carbon loss and microbial stabilization of substrates during

vermicomposting depended on rate of manure application



The decomposition systems depend on inputs of resources, that is, the components of the system do

not have any control on their availability (regeneration). Vermicomposting is the process whereby

organic residues are broken down by earthworms and microorganisms. Addition of manure has been

shown to be of critical importance and determines most of the changes that take place during

vermicomposting. Here we study how the rate of manure applied affects microbial biomass and

activity and carbon losses. Five samples of substrate per module were taken at random and gently

mixed for biochemical analyses, i.e. organic matter content, total C, microbial biomass C and basal

respiration. We found that earthworms increased microbial biomass and were more active in reactors

fed with 3 kg of slurry. However the differential rates of respiration were not reflected in C losses.

The results thus showed that loss of C was not affected by the rate of pig slurry applied. We conclude

that despite the strong effect that the rate of manure has on microbe-earthworm relationships, it did

not affect carbon losses. We therefore recommend the use of low application rates of manure when

the objective is the microbial stabilization of the residue.

281


5th to 10



Earthworms enhanced microbial populations triggering enzymatic

activities during vermicomposting of pig slurry



We assessed whether there is a relationship between earthworm activity, microbial biomass and the

activation and dynamics of several enzyme activities during vermicomposting. Since decomposition

systems are donor controlled we also study the effect of different rates of application of manure. For

this, we carried out an experiment in which low and high rates (1.5 and 3 kg respectively) of pig

slurry were applied to small scale reactors with and without earthworms. Five samples of substrate

per module were taken at random and gently mixed for biochemical analyses, i.e. microbial biomass

C (Cmic), microbial activity (basal respiration), β-glucosidase, cellulose, alkaline phosphatase and

protease activities. In both rates of pig slurry applied, earthworms stimulated microbial growth which

decreased once earthworms left the slurry. This increase was related to the initial activation of the

microbial enzymes studied: β-glucosidase, cellulase, alkaline phosphatase and protease as

correlations between microbial biomass and activity and enzymes showed. These strong relationships

indicated an increase of intracellular enzyme activity. In the aged slurry, the pattern of activity of the

four enzymes assayed depended on the rate of pig slurry applied. Thus, in low rate reactors,

enzymatic activity through layers appeared to be related to microbial biomass, but in high rate

reactors the activity of enzymes was more or less continuous. Further, these differences in overall

enzyme activity agree with the variation found in extracellular enzyme activity suggesting certain

dependence on substrate availability. We found that extracellular enzyme activity increased with rate

of pig slurry, indicating a baseline of enzyme activity through decomposition. Thus, rate of pig slurry

should be chosen carefully if vermicompost is going to be used as organic amendment, since

earthworm activity did not decreased it.

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Changes in structure and function of microbial communities improve

decomposition during vermicomposting of pig manure



Although microorganisms are largely responsible of organic matter decomposition during

vermicomposting, earthworms may also affect to rates of decomposition directly by feeding on and

digesting organic matter and microorganisms, or indirectly affect them through their interactions with

the microorganisms, basically involving stimulation or depression of the microbial populations. We

tested the general hypothesis that microbial populations, and especially fungi, are enhanced by

earthworm activity, and also whether earthworms are able to modify the biodiversity of microbial

populations, and its relation with the function of the system. In addition we examined the metabolic

quotient and the effect of labile organic C to assess the relationships between earthworm and

microbes. Five samples of substrate per module were taken at random and gently mixed for

biochemical analyses, i.e. microbial biomass-C (Cmic), ergosterol content, basal and substrate

induced respiration and Biolog® Ecoplate analysis. We found that decomposition of pig manure has

two stages characterized by the presence or absence of earthworms. Thus, the presence of

earthworms was related with increases in overall microbial biomass and activity which decreased

when earthworms left the substrate; the same pattern was observed for fungi. Furthermore,

earthworms modified the physiological profiles of microbial communities of pig manure, increasing

the diversity of substrates utilized. In addition, earthworms promoted a more efficient use of energy

of microbial communities as the metabolic quotient showed. The rate of carbon loss was almost twice

in the presence of earthworms, revealing faster decomposition. Our data match with the recent

findings that for maintaining essential processes the functional properties of present species are at

least as important as the number of species per se. This is in accordance with the ―insurance

hypothesis‖ which states that a large number of species is probably essential for maintaining stable

processes in changing environments, as presence of earthworms would have promoted in pig manure.

283


5th to 10



The Mexican Standard of vermicompost; scope and limitations

Aranda Delgado Eduardo1, Barois Isabelle

2, Velasquez Elena

3, Brunet Didier

4,

Lavelle Patrick4.

1 Terranova Lombricultores, Xalapa, Ver. Mexico 2 Instituto de Ecologia, Xalapa, Ver. Mexico

3 Universidad Nacional de Colombia, Palmira, Colombia

4 IRD, France

Since July 2005, the "Humus Working Group" was formed, to reach the formal document of the

Mexican Standard NMX-FF-109-SCFI-2008, published on June 10th

, 2008. Regular meetings were

conducted to establish the quality specifications to be met by vermicompost (solid state only) that is

produced or marketed in Mexican territory. These meetings were attended by earthworm cast

producers, researchers, associations and government institutions dedicated to the standardization and

certification, all coordinated by the National Standardization Technical Committee of Agricultural

Products, Livestock and Forestry, belonging to the SAGARPA (Ministry of Agriculture, Livestock,

Rural Development, Fisheries and Food). The group established the initial references, the objectives

and specific areas of application, definitions, descriptions and classifications of product quality. This

was defined by sensorial (smell and color) and physicochemical specifications, including the

presence of impurities, not digested and foreign materials, seeds and live earthworms. The

parameters selected to verify their quality were: pH, electrical conductivity, water content, ashes,

organic matter content, organic carbon, total nitrogen, C/N ratio, cation exchange capacity and bulk

density. Not even with the existence of all these test and specifications, it cannot be assured, -without

the possibility of error-, the certainty that some material is actually vermicompost; that means,

without no doubt, the material processed by earthworms. Trying to assure this indisputable certainty,

some representative samples of compost and vermicompost from different organic sources were

requested to test them with the Near Infrared Reflectance Spectroscopy methodology, NIRS. This

work showed the great potential of using this methodology, because apparently, it was not only able

to recognized the difference between compost and vermicompost, but it was also able to discern the

origin of organic matter used to feed the worms, even in mixtures. However, it remains to do more

comprehensive and complete studies, using more samples from different organic materials, paired on

both processing techniques, -with or without earthworms-, to discriminate those NIRS wavelength

values (nm) characteristic of the earthworm cast (earthworm signature) and perhaps, some other

useful quality or quantity organic matter parameters.

284


5th to 10



Consolidation of the Vermicomposting Technique at the Organic Solid

Waste Program in the Municipality of Teocelo

Barois Isabelle1, Cerdán Maria Angélica

2, Aranda Delgado Eduardo

3

Alarcón Gutiérrez Enrique 4

, Martínez T. Francisco5, Gonzalez C. Korina

2,

Santos R. Adriana1, Lopez C. Magdalena

1

1 Instituto de Ecología, A.C., Xalapa, Ver., Mexico,

2 Instituto Tecnologico Superior de Xalapa, Xalapa, Ver., Mexico,

3 Terranova Lombricultores, Xalapa, Ver., Mexico,

4 Instituto Nacional de Investigaciones Forestales, Xalapa, Ver., Mexico,

5 Director de Ecología y Desarrollo Rural del Mpio. de Teocelo, Ver., Mexico.

Teocelo municipality is located 15 miles from Xalapa, the state capital of Veracruz, in the coffee

region of the state's central highlands. Teocelo has a population of 14,900 inhabitants, 9,062 of which

live in the municipal center and the rest in 20 rural towns scattered in a vicinity of 21 square miles.

From 2001 to 2003 and from December 2008 till now, the municipality took the alternative to

operate a vermicomposting module to transform organic wastes into fertilizer. Daily, approximately 7

tons of agricultural, agro-industrial, forest and domestic organic waste are collected, transformed into

vermicompost and distributed among local producers through agricultural promoting programs. The

challenge of this program is not only the organic conversion by the worms, but it also consist to

know how to combine low cost environmental technology, citizenship participation and economic

development promotion. Up to now these are poorly articulated in local government policies in

Mexico. Although there are very little implementation of equipment, techniques and methodologies

which are rudimentary and little systematized, besides the difficulty to modify behaviour of the

public´s patterns for solid waste separation, the result of the vermicomposting treatment are

comparatively improved and process costs are lowered to the half. At this level of municipal

implementation, a multidisciplinary group has been formed, in order to technify the vermicomposting

treatment, to rationalize and improve the general operational methodology, implementing quality

control standards and build a procedure´s manual for a wider application, in other municipal solid-

waste processing facilities. Beyond the consolidation of the vermicomposting technique and the

regional impact of the program, the scope of the multidisciplinary group, is to replicate and escalate

many more processing facilities, to many other rural, small size municipalities in Mexico, which

have equivalent economic constraints; thus discouraging the expansion of sanitary landfills, toward a

better, cost-effective and environmentally-beneficial solution. This project has the financial support

of the National and State Council of Science and Technology, (CONACYT and COVECYT) with the

Fordecyt fund.

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Solid Residues management in the Faculty of biology of the

Universidad Veracruzana

De la Cruz Elizondo Yadeneyro, García Hernández Gabriel, Sosa Capistrán Dulce María,

Guzmán Jacob Valeria, Rodríguez Dioni Abril

Facultad de Biología, Xalapa, Universidad Veracruzana

The Biology Faculty of the Universidad Veracruzana, from Xalapa, produces a great quantity of

wastes which are separated by a Management Program of Solid Residues (RESU, from its name in

Spanish: Manejo de Residuos Sólidos Universitarios), that takes advantage of some recyclable

materials. Nevertheless, the organic matter generated (approximately 60% ) is comminuted to the

city's sanitary landfill. According to the Universidad Veracruzana‘s ideology on social development

and educational role, which can be found in the ‗Programa General de Desarrollo 2025‘, thus the

University is convinced of the prevailing necessity in Veracruz to generate and promote actions

toward sustainability and a wider usage of some resources that may represent a problem to the

environment and humans, within this statements is how this initiative emerges. In the beginning, this

workgroup was formed by students from the Edaphology course and has been growing by the

addition of new members from different generations; the vermicomposting station was established in

an area of 80 m2 and the process started with 20,000 worms of three different species: Perionyx

excavatus, Eisenia fetida y Eisenia andrei. Nowadays we get to treat most of the compostable wastes

produced within the Faculty, plus some other residues brought by voluntary donators and we are

ready to treat all wastes generated in mean building.Project‘s final goal can be divided in two parts,

first, diminish the environmental impact that our activities produce while we are inside the school

and; second, transmit among students the vermicomposting techniques we have learn, along with

some ideas about the importance and the advantages that solid wastes treatment can offer. The same

working space has led the creation of a new area for research and practice within the school. We have

achieved to create a working and learning space where new projects are beginning to take form:

Urban agriculture systems, organoponia and acuaponia are some examples, besides vermicomposting

have awakened the interest and the desire in many of our students to participate in this project

through social service, while at the same time it encourages the collaboration between the students

and faculties in the Universidad Veracruzana.

286


5th to 10



Physicochemical characterization of vermicompost from rumen content

and hen manure

Flores Magallon Rebeca, Caraveo Dominguez José Manuel, Alvarez Bernal Dioselina,

Sanchez Nuñez Minerva, Herrera Arreola Guillermo

CIIDIR-IPN, Unidad Michoacán, Mexico.

Handle and final disposition from solid residuals generated by municipal slaughterhouses (rumen

content) and hen manure generally do not receive a suitable treatment; therefore causes a very serious

contamination problem in the environment and life quality of human being. The aim of this study

was to produce vermicompost using Eisenia foetida for recycling rumen content and hen manure, as

well as to characterize physicochemically both vermicomposted. In order to do recycling process;

eight beds settled down, four were for recycling rumen content and the rest for hen manure, the beds

had the following measures: three meters in length by one meter in width. The physicochemical

characterization of the vermicompost was made according to the methodologies recommended by the

NMX-FF-109-SCFI-2008 (Mexican Standard). Both substrates were stabilized in a lapse of 45 days.

The vermicomposting process is a good alternative for handle and disposition of slaughterhouses

residuals and hen manure from farms as long as it is fulfilled four fundamental requirements: a)

Superficial and underground waters are protected of contamination, b) reduce the risk of propagation

of animal diseases, c) avoid de prescence of flies, larvae, noxious fauna, etc. and c) keep the air

without of bad odors. Vermicomposting is a simple, economic and very effective procedure for

recycling organic residuals. We obtained a mature and stable vermicompost, we can conclude that

vermicompost from rumen content and hen manure can be used as biofertilizer or to enhance

degraded soils.

287


5th to 10



Ecofriendly Treatment of Biomedical waste Using Epigeic earthworms

Geetha K.S. 1, Dinesh M.S.

1, Chandana P.G.

1, Kale Radha D.

2

1 Department of Biotechnology, PES Institute of Technology, India,

2 Center for Scientific Research and Advanced Learning, Mount Carmel College, India.

Hospital wastes are hazardous and need to be disinfected before disposal. Biomedical waste (BMW)

contains spilled liquid orals, antibiotics in wound dressing and tissues resulted during surgery

constitute 30% of the urban waste (Walsh et al 2000). Land filling and incineration are the present

methods. Former method leading to soil pollution and the latter is an expensive and energy

consuming process. The present study was conceived using earthworms Eisenia fetida, Eudrilus

eugeniae, and Perionyx excavatus to compost the bio-degradable part of infected BMW. The Bio

Medical Waste was primarily treated with 5% NaCl for chemical sterilization process and the same

was later subjected for detection of pathogens by pour plate method with specific media. The

pathogens analysed in the BMW were Escherichia Coli, Staphylococcus aureus, Salmonella typhii,

Pseudomonas auriginosa, Bacillus cereus, Clostridium sp., and Klebsiella. They were in the order of

2x106 cfu/g using bio-rad microbial colony counter. Initial processing of the waste was done by

adding cow dung slurry in 1:1 ratio. A biomass of 200g of each species of earthworms was released

into the containers filled with 5kg of waste and a control was maintained without earthworm. After

30 days of composting period, increase in earthworm biomass, compost yield and pathogen load in

the compost were analysed. The study revealed that Eisenia fetida exhibited better performance in

conversion of BMW with 40% increase in biomass with 60% compost yield. The biomass increase in

Perionyx excavatus was 25% with 45% compost yield and in Eudrilus eugeniae, it was 15% increase

in biomass with 40% compost yield. It is clear from the results that Eisenia fetida is more suitable

species for composting of BMW than Perionyx excavatus and Eudrilus eugeniae. There was decrease

in pathogen level when compared to control during composting compare to control from 2x106 cfu/g

to 1x102cfu/g. The composted material is subjected storage in polythene bags. There is no further

increase in pathogens level inferring the ability of Vermicompost in suppressing the growth of

pathogens of BMW.

288


5th to 10



Biomanagement of sugar factory press mud through the application of

vermitechnology and its application on black gram

Gobi Muthukaruppan, Gunasekaran Paramasamy, Vijayalakshmi G.S.

Dept. of Genetics, School of Biological Sciences, Madurai Kamaraj Univ., Tamilnadu, India.

Vermicomposting of sugar factory pressmud using two different species of earthworm via., Eudrilus

eugeniae and Lampito mauritii was prepared. Plant nutrient contents of these vermicompost and their

effects on black gram (Vigno mungo) growth in relation to the effects of sole soil were investigated.

Vermicompost produced by the two species of earthworms differed in their nutrient concentrations.

But possessed higher concentration of total N and Ca than that of the control. The results showed that

effects of vermicompost are more efficient for the vigorous production of black gram. Vermicompost

had significant positive effects on flower number, leaf growth, shoot length, root length, number of

leaves, leaf area index, wet weights and dry weights compared to control. It is also suggested that

pressmud compost is more favourable for vigorous production of black gram and maintenance of soil

environment but vermicompost can be economically and environmentally suitable.

289


5th to 10



Earthworms modify microbial community structure and activity and

accelerate decomposition during vermicomposting

Gómez Brandón Maria1, Aira Manuel

1, Lores Aguín Marta

2, Domínguez Jorge

1

1 Universidad de Vigo, Spain,

2 Universidad de Santiago de Compostela, Spain.

The vermicomposting process includes two different phases regarding the activity of earthworms, (i)

an active phase during which earthworms process the organic waste, thereby modifying its physical

state and microbial composition, and (ii) a maturation-like phase marked by the displacement of the

earthworms towards fresher layers of undigested waste, during which the microbes take over the

decomposition of the earthworm‘s processed waste. In the present study we investigated the direct

and indirect effects of the epigeic earthworm species Eisenia fetida on the microbial community

structure (bacterial and fungal PLFA biomarkers) and activity (basal respiration), and on organic

matter decomposition (total and dissolved organic carbon content) during the vermicomposting

process of pig slurry. We set up a batch of twelve reactors, six with a low dose of pig slurry and the

other six with a high dose. For each dose, three reactors were inoculated with 500 mature specimens

of E. fetida and three remained without earthworms (control). Each reactor was initially composed of

one module containing vermicompost from pig slurry, in which earthworms were placed, and another

module containing a layer of fresh pig slurry (1.5 or 3 kg fresh weight, low and high rate,

respectively). New modules with the same amount of fresh pig slurry were added sequentially

following the feeding activity of the earthworm population. At the end of the experiment, the reactors

comprised 12 modules with an increasing gradient of age, resembling a soil profile, from upper to

lower layers as follows: 2, 4, 7, 8, 11, 18, 21, 25, 27, 29, 33 and 36 weeks. The principal component

analysis of the 25 identified PLFAs clearly distinguished between samples in function of earthworm

presence and age of layers in both high and low rate reactors. The activity of epigeic earthworms

reduced the abundance of bacterial and fungal PLFA biomarkers relative to the control during

vermicomposting; although their impact on bacterial populations was more pronounced in low than

in high rate reactors, probably due the fact that the greatest losses of carbon as a result of earthworm

activity occurred in the reactors fed with 1.5 kg of pig slurry. These findings indicate that

detritivorous earthworms play a key role in the decomposition of organic matter during

vermicomposting via their intense interactions with decomposer communities, which may have

important implications for the stabilization of the organic residue.

290


5th to 10



Bulb formation in Lilium sp from hypogeous bulblets grown in different

proportions of vermicompost

González Nicanor Araceli, Grenon Cascales Graciela Noemi,

González Castellanos Anacleto, Perez Hernandez Amalia

UAEM, Mexico.

There are a technological dependence of Mexico in the ornamental horticulture, especially in the

acquisition of vegetative material; thus is needed research of appropriate, sustainable technologies

that allow to obtain good quality bulbs, adapted for satisfactory crops, specially for producers with

low input resources. In the majority of the cases these producers do not have access to imported

materials, high cost technology, surely resulting in good quality vegetative materials. The formation

of bulbs of Lilium sp. with a technology adapted to zones and low input resources, does not imply the

violation of rights of possessing one, since this technology is realized by themselves, cultivating

free-access varietes, not protected by the rights of possessing. In this context , the aim of the present

work was the production of bulbs of Lilium sp. from hypogeal bulblets grown in different proportions

of vermicompost. The work was realized in the Faculty of Agricultural Sciences of the UAEM, in

greenhouse conditions; we can conclude that the best depth of sowing the hypogeal bulblets is around

15 cm., the best time of refrigeration to induce vernalitation in the hypogeal bulblets is around 90

days at 2°C. The mixture formed by 60 % of vermicompost, 10 % litter soil and 30 % perlite was the

best option in terms of leaf area , fresh weight and length for the initial bulb.

291


5th to 10



Effects of biological conversion on winery waste using Eisenia foetida

Hlavova Andrea, Landa Ivan, Mikulova Vlastimila

Czech University of Life Sciences, Prague, Faculty of Environmental Sciences, Kamycka 1176, Prague, Czech

Republic.

Our observations and literature search have shown that vermicomposting is a very suitable method

for utilization of waste from wine production. This organic mater is usually used as an agricultural

fertilizer, however, overproduction of grape pomace from wine production in recent years has led to

its direct use in rural areas and due to the release of tannins and phenols, this trend has resulted

negative effects on growth of plant roots. Not only this negative effect of tannins, but also some of

adverse qualities of matter could be reduced by the treatment of wine waste using biological

convertion of matter (vermicomposting) as an alternative to their disposal or use for direct

application. The paper summarizes results of experiments aimed at utilization of wine waste using

Eisenia foetida in laboratory and outdoor conditions. There are described also technological aspects

of the process of biological conversion and main significant changes in product quality, especially

increase of the agronomic value, reducing of the C:N ratio, increase of pH, content of nutrients and

higher microbiological activity.

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Session 8 - Poster

Production of vermicompost with organic wastes in 50 homegardens of

Tabasco, Mexico

Huerta Esperanza, Flores Lorena, Van der Walls Hans, Ponce Alejandro, Magaña Mauricio,

Javier Sigrid, Jarquin Aaron

El Colegio de la Frontera Sur, Unidad Villahermosa

In Tabasco, southeast of Mexico, it exists an increasing problem of wastes production, even in the

traditional homegardens we observed that wastes are not well managed, they are put in corners of the

homegardens and sometimes they are incorporated into the soil: metal, plastic and organic matter

together, or they are just part of the landscape as little hills of garbage. As a part of a

multidisciplinary project we installed into the 5 physiographic regions of the state of Tabasco, inside

of 50 homegardens, systems for producing vermicompost, these systems were constituted by metal

boxes of different sizes: 14 boxes of 40x50 cm and 36 boxes of 20x50 cm, with a mill inside, with

the earthworm species Eisenia foetida, wastes were introduced into the systems once they have been

produced and within 18 days the vermicompost was produced. The mountain region had the highest

production of wastes: 0.8/day/per person, being the organic matter the 56% of the total garbage. 44%

of all the vermicompost systems work successfully. More studies are required in order to observe the

effect of the vermicompost over the plants of the homegardens.

293


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Effect of Aloe vera composted residues on reproduction of Eisenia foetida

Imery Buiza José

Depto. de Biología, Universidad de Oriente, Venezuela.

Aloe vera is a succulent plant introduced to America and widely cultivated for medicinal uses. Aloe

industry that processes the leaves and remove the gel, which made cosmetics and therapeutic drinks,

discard the mesophyll (M) enveloping the hydroparenchyma, representing almost 30% of wasted

tissue. Moreover, on large plantations with low expansion, separation of suckers (S) from mother

plants, is also a vegetal biomass that is lost if they are not planted in new areas. The aim of this work

was to evaluate the reproductive performance of the Californian red worm (Eisenia foetida)

experimentally raised in different composts made from a mixture of horse manure (HM), dry chaff

(DC) of Phragmites australis and aloe fresh residues (M and S). Preliminary composting lasted 90

days, using heaping with roll over method. Multiplication rate (MR) of earthworms (EW) was

evaluated, set initially at a density of 15 EW/L in plastic crates of 40 L. Treatments were established:

T1) composting of the mixture 90% HM + 10% DC; T2) 60% HM + 30% M + 10% DC; T3) 30%

HM + 60% M + 10% DC; T4) 60 % HM + 30% S + 10% DC; T5) 30% HM + 60% S + 10% DC,

with five repetitions, humidity 65-80% and temperature 22-26 ºC. The experiment ended after 120

days, and significant differences between treatments (ANOVA).

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Growth and reproduction of the earthworm, Perionyx ceylanensis Mich.

during vermicomposting of different substrates

Jayakumar M. 1, Karmegam Natchimuthu

2

1 Dept. of Chemical Eng., Arulmigu Meenakshi Amman College of Engineering, Tamil Nadu, India,

2 Dept. of Biotechnology, VMKV Engineering College, Tamil Nadu, India.

It is absolutely essential to study the growth and development of earthworms in order to promote

vermiculture based biotechnology. This may help in understanding the organism, selecting prolific

breeders, predicting the population characteristics. Perionyx ceylanensis Mich. is a purple–red

coloured epigeic earthworm, mainly found in biogas slurry, dung pats, composting heaps and

decomposing leaf litter heaps and has been recently reported for its efficiency in vermicomposting.

But its growth and reproduction during vermiconversion of organic materials is not studied in detail.

Hence the present study has been carried out to assess the growth and reproduction of P. ceylanensis

during vermicomposting of pressmud, pressmud+leaf litter, cowdung and cowdung+leaf litter. trials

in 45 × 35 × 15 cm plastic troughs containing 2 kg of substrates. The vermibeds were maintained at

27±1°C with a moisture content of 70±5%. Sixty clitellate P. ceylanensis, were placed in each trough

covered with wire mesh. The cocoons were collected from the vermibeds after 10th of inoculation up

to 60 days and the worms were weighed at the termination of study on 60th day. The highest

individual biomass of 780 mg/worm was recorded in pressmud followed by cowdung and

cowdung+leaf litter (1:1). The cocoon production rate of 0.95, 0.98, 0.89 and 0.92 cocoon/worm/day

was observed in pressmud, cowdung, pressmud+leaf litter and cowdung+leaf litter. The present study

shows that the earthworm species, P. ceylanensis is a prolific breeder comparable to that of P.

excavatus.

295


5th to 10



Eradicating flies (Diptera) in earthworm ecological boxes

Kostecka Joanna, Garczyska Mariola

University of Rzeszow, Faculty of Agriculture and Biology, Rzeszow, Poland.

A consumerist lifestyle generates great amount of waste, including organic waste. One of the ways to

manage it on-site – in a place it is produced, is ―earthworm ecological box‖. Vermicomposting in

those small containers can be problematic as relatively small earthworm population loses its

dynamics of growth easier in ecological boxes, it can also become dominated by competing

Enchytraeidae and, what is more uncomfortable to operate, flies Sciaridae can proliferate in

ecological boxes. The aim of the experiment, which concerned bio-preparation ―Owinema‖ and a

xenobiotic DAR 2.5 GR (active substance: chlorfenvinphos), was to observe the impact of the

products eradicating flies larvae on the state of earthworm Eisenia fetida (Sav.) which

vermicomposted (the same amount and quality) of kitchen waste. The doses of the preparations used

were suggested by the producer. Before experiment the number of flies had been increased (by

multiplying it for 4 weeks on mushroom mycelium infected with larvae of Sciaridae). In each of 20

containers (capacity of 2 dcm3) 50 earthworms were bred for 5 months. The dynamics of the

population properties was being checked on regular basis by hand sorting. The rate of

vermicomposting and the quality of the fertilizers produced were also determined. It was observed

that the bio-preparation ‖Owinema‖ had a positive impact on the average number and biomass of

earthworms when compared to the control.

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Short term effects of vermicompost on soil microbial and biochemical

properties in a sweet corn (Zea mays) crop

Lazcano Cristina1, Gómez Brandón Maria

2, González Porto Pablo

2,

Domínguez Jorge 2

1 Centro Tecnológico del Mar - Fundación CETMAR, Spain,

2 Departamento de Ecología y Biología Animal, Universidad de Vigo, Spain.

Organic fertilizers have shown to improve significantly the soil status through the amelioration of its

physical and biological properties. Therefore they allow an increase in soil fertility which could

result in better crop growth or quality. However these changes are normally produced in the long

term therefore questioning the profitability of this type of amendments in the short term as compared

to synthetic mineral fertilizers. The short-term effects of the partial substitution of mineral fertilizers

by vermicompost on the soil biological properties of a sweet corn crop were evaluated in a field

experiment. The effects of the equivalent non-processed organic matter (manure) were also evaluated

in order to determine if earthworm processing produced any biological gain in the fertilizing capacity

of this material. Full inorganic fertilization was used for comparison and two doses (normal:

80/24/20 and high: 120/36/30) of the three fertilizing treatments were added. Experimental units

consisted of 10m2 plots each with one combination of fertilizer and dose. Plots were overplanted and

thinned to obtain a final density of approximately 60000 plants ha-1, and arranged in the field

following a randomized block design. At harvest, three months after sowing, the concentration of

available plant macronutrients (N-NH4, N-NO3, K2O, PO4), microbial community structure (PLFAs),

and activity (protease, phosphatase and glucosidase activity, basal respiration), were evaluated in soil

samples collected from the plots. Macronutrient content was similar among the different fertilizing

treatments. Significant differences were only observed in the case of PO4 which was significantly

higher in vermicompost plots. Partial substitution of mineral fertilizers by vermicompost produced

also significant changes in the structure of the microbial community as evidenced by the discriminant

analysis of the phospholipid fatty acid (PLFA) profiles of the soil samples. Changes were due to an

increase in gram negative bacteria with vermicompost and manure as compared to full mineral

fertilization, especially in the case of manure. These changes were accompanied by an increase in

soil metabolic activity with significant increases in phosphatase and glucosidase activities with

vermicompost and protease activity with both organic fertilizers. The integrated use of organic and

inorganic fertilizers significantly improved the soil biological properties as compared with full

inorganic fertilization while maintaining the concentration of available plant macronutrients. Effects

were observed in a three month growing period showing that these types of practices do not risk crop

productivity and allow a short-term amelioration of soil properties. Vermicompost enhanced the

release of PO4 although no other beneficial effects were observed as compared to manure.

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Assessment of fertilizer power of vermicompost obtained by utilization of

municipal slaughterhouse wastes

López López Alberto, Gallardo Valdez Juan, Contreras Ramos Silvia Maribel

CIATEJ, Mexico.

The common problem in municipal slaughterhouses in Mexico is the absence of processes for

management and final disposal of solids and liquid wastes. Approximately 20% of the total animal

weigh slaughtered are solid wastes, such as rumen, manure and liming. Today, these are not used for

food consumption and have no commercial value. The aim of this research was to assess the fertilizer

power of a vermicompost and leachate obtained from slaughterhouse waste. The rumen and manure

of slaughterhouse were mixed with vegetable wastes and pre-composted for eight days. The

vermicomposting process was performed in a laboratory with 8 kg of total wastes and 0.5 kg of

Eisenia fetida earthworms (200 individual m-2 density) with four replicates during two months.

Vermicompost and leachates from the process were recovered and characterized by physicochemical

parameters, microbiological and phytotoxicity tests. An experimental design (32) in triplicate was

used to investigate the vermicompost and leachate fertilizer power with a tomato crop in a

greenhouse during three months. These were compared with a commercial fertilizer. Three dose

levels of fertilization were used: high (300 kg N·ha-1

), medium (200 kg N ·ha-1

) and low (100 kg N

·ha-1

). Height, number of leaves, chlorophyll, photosynthesis, total leaf area and total weight of fruit

were evaluated. The yield of transformation of waste by vermicomposting process was from 55% to

65% and 1.9 L of leachate was recovered. The N/C, P/C K/C ratios of waste have a direct

relationship with fertilizer power from the vermicompost and leachate. Those have a significant

effect on plant growth and yield from a tomato crop. The vermicomposting process was successful in

converting the municipal slaughterhouse waste. The vermicompost and leachate obtained had

potential characteristics for use as organic fertilizer.

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Characterization of Organic Alternative Substrates for the Production Of

Potato Tuber- Seed

Patrón I. José Cinco1, Alcántar G.G.,

2, Quintero L. R.

2, Pineda P. J.

3, Sandoval V. M.

2,

Brown George 4

1 Depto. de Agroecología y Medioambiente, Inst. de Ciencias, BUAP, Mexico,

2 Programa de Edafología, CP-Montecillo, Mexico,

3 Depto. de Suelos, UACH, Mexico,

4 EMBRAPA Florestas, Curitiba, Brasil.

Mixtures of organic and inorganic substrates for food production in greenhouses need to be evaluated

physically, chemically and agriculturally in order to know their properties and thereby adjust the

ferti-irrigation system. Thus to be used in intensive production systems as the seed potato production

in greenhouses. We evaluated six mixtures of substrates by replacing only the peat component of the

mixture used traditionally by producers, which consists of 40% coir, 40% peat and 20% perlite, all in

a relationship v/v, materials. The substrates to test were peat (control), pine sawdust, rice hulls,

compost from agave tequila, vermicompost from coffee pulp and sugar cane bagasse. The potato

varieties used were Alpha and Atlantic. The substrate mixtures were placed in beds divided into

squares 0.5 m x 0.5 m, with a height of 15 cm and a density 69 plants . m

-2. Each treatment had four

seed tubers, with 12 repetitions, having a total of 144 experimental units l, 288 tubers of each variety

wasused. The results indicate that the largest number of mini-tubers per plant in the Alpha range

were obtained in the screening substrate mixture of coffee pulp (9.5) was statistically peat treatment

(control) 5.75 and for the variety Atlantic sieving treatments coffee pulp (5.9), compost of agave

tequila (4.7), peat (4.4) and rice husk (4.4). The aeration capacity (AC) was higher in treatments with

15.9% sawdust, coffee pulp screening (15.8%) and rice bran with 12.8%. In the total pore space

(which is the sum capacity of aeration and moisture holding capacity) processing peat (76.0%) and

bagasse from sugar cane (75.3%), where the latter is not allowed in irrigation water beginning of the

experiment (hydrophobicity), and this present compaction. On both Alpha and Atlantic range

submitted diseased tubers with common scab (Streptomyces scabies), although with a low incidence

per plant.

Acknowledgements to the owner Engineer Manuel Villarreal Galicia, Agrícola Villarreal Company,

producers of pre-basic seed potatoes, for the facilities granted to carry out this work in the

greenhouse, also to M.C. Joel Pineda Pineda responsible for the laboratory area of Plant Nutrition

Soil Fertility Universidad Autonoma Chapingo, for allowing the physical and chemical

characterization of the substrates and CONACyT Mexico for the financial support.

299


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Quality of humus produced by Eisenia andrei from three organic

substrates. Utility in agriculture and ecotoxicology

Polo Aldo, Marcano Leida, Martínez Rosa, Marcano Elena, Hernández Juany,

Cortesía Adriana

Universidad de Oriente, Venezuela.

This study examined physical chemical characteristics of humus produced by earthworm Eisenia

andrei from three organic substrates prepared with a mixture of horse manure (HM), pruning wastes

(PW) and kitchen wastes (KW) in the following treatments: T1 (60% HM + 40% KW), T2 (60% HM

+ 20% KW + 20% PW) and T3 (60% HM + 40% PW). E. andrei was used due to their biological

characteristics conducive to biotransformation of organic wastes into fertilizer. Humus samples were

sieved (that humus produced from these wastes have high levels of macro and micronutrients and

heavy metals concentrations below the limits set by environmental legislation, making it an optimal

choice for fertilizing crops.

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Bio-remedial measures of a monocot weed, Saccharum munja using

Eisenia fetida and impact of vermicompost on the shoot length

of Vigna radiata

Shing Satyendra M. , Gangwar Geeta R., Ajay Neha

Vermiculture & Environmental Research Lab., Dept. of Animal Science, Mahatma Jyotiba Phule Rohilkhand

Univ., India.

Saccharum munja (munj) – a cosmopolitan weed, is known to develop barren conditions in the soil,

has been taken for its bio remedial measures by inoculating epigeic worm, Eisenia fetida. Weed was

collected from nearby University area, chopped into small pieces of 2 cm size. The cut pieces were

mixed with cattle dung in 1:3, 1:1 and 3:1 ratio separately. The experimental sets of each ratio were

prepared in triplicate in rectangular plastic tubs, each having weight of 4 kg. The control sets, devoid

of earthworms, were also put aside with respective experimental sets. Ten g matured E. fetida were

introduced in each of the experimental sets after 15 days of pre-decomposition period. Physico-

chemical parameters of weed: dung media and vermicompost were analysed by using standard

techniques. The experiment was conducted for successive two years. It was noticed that 1:3 weed:

dung medium transformed earlier into vermicompost than that of other two media (1:1 and 3:1). Rate

of reproduction of worms was recorded 2.47 times faster and their total biomass was 2.15 times more

in 1:3 medium than that of 3:1. Vermicompost transformed from 1:3 weed: dung medium when used

with soil in the ratio of 1:1 showed excellent results in the shoot length (26.0 cm) of pulse plants,

Vigna radiata, although increase in shoot length (15.5 cm) was also noticed when vermicompost

transformed from 3:1 weed: dung medium was used with the similar ratio of soil. It may be

concluded that by using epigeic earthworms, the weed may easily be transformed into biomanure that

could be used in improving soil health for the better crop yield.

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Effect of the type and proportion of physical conditioners on the biology of

earthworms and chemical properties of the humus

Schiedeck Gustavo1, De Almeida Schiavon Greice

2, Priebe Holz Fabiana

3,

Ernani Schwengber José1

1 Embrapa Clima Temperado, Brasil,

2 Universidade Federal de Pelotas, Brasil, 3 Instituto Federal Sul-rio-grandense, Brasil.

The use of crop residues with high C/N ratio mixed with the food offered to the earthworms can be

useful to improve environment drainage and aeration, promoting the earthworm development and

reproduction of the. The objective of this work was to evaluate the effect of two materials for

physical conditioners of the food on biological aspects of earthworms and their impact on the

chemical properties of the produced humus. The study was conducted at the ―Estação Experimental

Cascata‖, ―Embrapa Clima Temperado‖, in Pelotas, RS, Brazil, using cattle manure as the main food

and oat straw chopped and crushed peanut hulls as physical conditioners. Plastics pots of 100 mm

diameter were filled with cattle manure (CM) and mixtures of CM with chopped oat straw (OS) or

crushed peanut hulls (PH), at 2:1 (OS33 and PH33) and 1:1 (OS50 and PH50 ) in volume/volume

ratios. In each pot were placed 10 adults of Eisenia andrei and weekly evaluated their biomass

progress, cocoon production and food volume evolution. After 68 days the chemical characteristics

of humus were analyzed. A completely randomized design, with five treatments and three replicates

was used. The means were compared with the Scott-Knott test at the probability level 5%. Physical

conditioners did not affect the biomass of earthworms. However, there was a tendency of the

earthworms in the OS50 treatment to produce larger biomass, and the PH50 treatment to produce

smaller biomass. The OS50 and CM treatments did not differ amongst themselves in relation to the

total average cocoon production, ranging between 300.52 and 285.26 cocoons, but they were

statistically higher than other treatments. The lowest value occurred in the PH50 treatment, with only

256.02 cocoons. The final volume of produced humus in the OS50 and OS33 treatments was 47.9%

and 52.4% of the original volume, respectively. PH33, PH50 and CM treatments did not differ

amongst themselves in relation to the final volume, ranging between 65.6% and 77% of the original

volume. PH33 and PH50 treatments showed smaller levels of N, P, K, Ca, and Mg, while OS33,

OS50, and CM had similar values amongst themselves. The results indicate that the use of chopped

oat straw in a 1:1 ratio by volume with cattle manure favours the reproduction of the earthworms

without affecting their growth and the chemical properties of humus.

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Production of biofertilizer (Rhizobium) enriched vermicompost from

pressmud using Eudrilus eugeniae and Perionyx ceylanensis

Sekar Kuppuraj Raja1, Karmegam Natchimuthu

2

1 Emu Faarms India Limited, Elachipalayam, Tamil Nadu, India,

2 Department of Biotechnology, VMKV Engineering College, Tamil Nadu, India.

In the present study, a trial has been made to prepare Rhizobium enriched vermicompost from a

sugar-industry by-product, pressmud so as to get dual benefit from a single product. Vermibeds were

prepared in plastic trays of 45×30×15 cm size with 3 kg of 20 days pre-decomposed pressmud in

each tray with a moisture content of 65-75%. After 24 hrs stabilization clitellate adult worms (30

numbers of Eudrilus eugeniae Kinb./trough and 60 numbers of Perionyx ceylanensis Mich./trough)

were introduced. Rhizobium leguminosarum (MTCC 99) procured from Microbial Type Culture

Collection (MTCC) Chandigarh was mass multiplied in Yeast Extract Mannitol Salt Agar (YEMA)

media and used for the study. R. leguminosarum at the rate of 30, 35 and 40 ml/100 g of substrate

was inoculated on 0, 10, 20 and 30 days of vermicomposting. Each experimental set-up was

maintained in triplicate. After 40 days of vermicomposting, the vermicompost was harvested and

analysed for total NPK, organic carbon, C/N ratio and total viable population of R. leguminosarum.

The viability count of R. leguminosarum in vermicompost was carried out by spread plate method

using YEMA medium. Once in 15 days, the vermicompost was analysed for the total viable count of

R. leguminosarum up to 75 days to assess the survivability upon storage. The C/N ratio of pressmud

vermicompost produced using E. eugeniae and P. ceylanensis was 13.4 and 14.2 respectively. The

total NPK contents of the vermicomposts produced by both the worms showed increase due to

mineralization. On 75th day of storage, the pressmud vermicompost of E. eugeniae showed 4, 7 and

6 CFU x 107

viable cells of R. leguminosarum respectively in 30, 35 and 40 ml/100 g inoculated on

0th day of vermicomposting. The inoculation of R. leguminosarum at the rate of 35 ml/100 g

substrate on 30th day of vermicomposting showed higher viable counts on 75th day of vermicompost

storage. The same trend has been observed in the pressmud vermicompost of P. ceylanensis. It is

concluded that the amount and time of inoculation of R. leguminosarum in vermicomposting systems

are important for the long term survival of the biofertilizer organisms to get multiple benefits when

the vermicompost is applied to soil.

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Preference of Eisenia foetida for vegetable waste of kitchen

Villegas García José Guadalupe, Rivera Zamora Raymundo, Covarrubias Prieto Jorge,

Raya Perez Juan Carlos, Alvarado Barcenas Estefana, Aguirre Mancilla César L.

Inst. Tecnológico de Roque, Celaya, Guanajuato, Mexico.

The use of earthworms is an alternative to organic waste management, especially in urban areas, in

addition to adding vermicompost to the soil they will not only improve in nutrient content but also in

structure. The objectives of this study were to quantify the acceptance of the California red

earthworm (Eisenia foetida) to bovine manure and organic waste from kitchen, both composted. The

bovine manure came from the livestock facility and the waste plant material from the dining room

both from Instituto Tecnologico de Roque. The treatments were: (I) composted manure EC, (II)

organic waste DO, (III) combination of EC + DO 1:1 proportion (IV) combination EC + DO 2:1

proportion and (V) combination EC + DO 3:1 proportion. Each treatment had four replicates, were

placed in plastic containers of 20 litres. The moisture content was 80%, the experimental units were

placed in a greenhouse and inoculated with the earthworm, 50 per experimental unit. The substrate

temperature was measured every day and checked that the humidity in the substrate was 80%. A

sample of substrate by repeated to quantify the density of earthworms, total biomass and was

determined the pH of substrate. The study was done with a design in complete blocks at random. The

population counts of young and adult earthworms were performed at 30, 60 and 90 days. According

to statistical analysis, highly significant differences exist for treatment. The Tukey test shows that the

treatment (II) DO, than the other treatments since it significantly increased the population of

earthworms. It was also noted that the number of cocoons exits differences in treatment, increasing

these one in the treatment II. Differences in pH and electric conductivity were detected in the

substrates, for treatment II the values of these variables were the lowest.

The completion of this work is due to funding provided by DGEST key 2704.09-P.

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An eco-engineering of integration of crop-vermiculture to treat organic

waste in situ

Yupeng Wu, Xiong Hui, Sun Zhenjun


We designed an intercropping system with vermiculture in the field to process organic waste in situ.

the study was done using winter-wheat, summer-maize rotation cropping system, with rows of

vermicomposting beds in between to transform organic wastes such as cow manure, municipal sludge

and mushroom residuals, comparing with conventional agriculture. We evaluate the effectiveness of

the model considering different organic wastes convertion, the crop production, the environmental

effects and the economic benefits. The results showed that the social and economic benefits of the

cropping-livestock system of corn/wheat- earthworm were higher than that of the traditional cropping

pattern of corn wheat: (1)The system is an effectively waste processing method; the waste reduction

and the nutrient content obtained was different because of different types of organic waste used.

Manure treatment not only get a 70% reduction but also get a more high-quality organic fertilizer,

which total nutrient reached 4.3%, in contrast to this values, ,mushroom waste treatment only obtain

a 55% reduction and 2.8% total nutrient content. (2) In this system, through the rational close

planting, we can get a higher corn yield than traditional, but a half when grow wheat because of the

decreased planting density. But the overall seed weight cropped, size of the grain, nitrogen,

phosphorus, potassium, protein content, is better than of the traditional. (3)In the harvest season, we

not only get corn or wheat, but also earthworm and organic fertilizer, with no chemical fertilizers

input and mechanical cultivation reduction, we get an increased revenue form crop-vermiculture

system than traditional agriculture. (4)The earthworm activity certainly improved the soil physical

structure, maintained and increased the soil nutrients, playing a fertililizer role. (5) We found that the

treatment with sludge -with available heavy metals, results in a slight enrichment of Zn content in

soil; In the harvested grain was also higher, but no more than the National Security Standard. In a

word, the organic waste produced on the farm was transformed by earthworms and at the same time,

kept a good soil quality for sustainable organic farming system. The cropping-livestock system could

not only improve the soil fertility and structure, but also controled the weeds in winter. Therefore,

there was a big decrease in amount of herbicides and pesticides, and the field ecology and

environment could be protected well.

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Interaction of vermicompost and peanut in iron mine tailing ecological

restoration: laboratory experiment

Zhang Junying, Xu Yongli, Li Fu-ping, Han Dong-yun

College of Ressource and Environment, Hebei Polytechnic University, China.

In order to improve poor fertility and microbial environment of iron mine tailing in its ecological

restoration, cow manure vermicompost was applicated into iron mine tailing, and peanut (Arachis

hypogaea) as restoration species was sown in mixture of iron mine tailing and vermicompost in

laboratory condition. Four application rates of cow manure vermicompost (in volume), 10%V(1:9),

20%V(2:8), 30%V(3:7), were designed in this experiment, and 0%(0:10) was control, CK. Three

peanut seeds were sown in each pot contained 1kg iron mine tailing or mixture of iron mine tailing

and cow manure vermicompost. Three replicates were tested for each treatment. Each pot was

watered with 150mL tap water every day. The germination, plant height, biomass of plant were

recorded during experiment. 35-day after germination the seedlings were harvested and three

nutrients elements, such as N, P, and K and microbial population, bacteria, fungi, actinomycetes, in

iron mine tailing were determined. The data showed that cow manure vermicompost stimulated

peanut seedlings height, root length, and dry weight of shoot. For example, plant heights grown in

pot with 10%, 20%, and 30% vermicompost were higher 83.3%, 67.6%, 63.5% than that of CK,

respectively. And seedlings root length and dry weight of shoot grown in pot with

vermicompost(10%, 20%, 30%) were longer 11.0%, 3.23%, and 2.91% and more 13.7%, 5.8%, -

6.8%, than those of CK, respectively. Peanut seedlings root exudate was beneficial for microbial

thrive. The population of bacteria in rhizosphere of 35-day seedlings was more than that of CK. The

sequence of bacteria population or fungi population in rhizosphere of four treatments was 10%V >

20%V > 30%V > CK. While the actinomycetes population sequence was 10%V > 30%V > 20%V >

CK. After peanut seedlings were harvested the nutrient contents, such as N, P, and K, increased than

before peanut planting. The increment percentages of N and P elements were 10%V(243.8% and

204.5%) > CK (100% and 172.2%) > 20%V(52.3% and 72.0%) > 30%V(-26.5% and 39.1%) as

comparing the value of mixture after seedlings harvested to that before planting, respectively. While

the K element increment percentage was CK(187.7%) > 10%V (106.2%) > 20%V(45.2%) >

30%V(23.4%). It was concluded that cow manure vermicompost stimulated peanut seedlings growth

as it was used into iron mine tailing during plant restoration. The 10% vermicompost in mixture was

an appropriate application for this experiment. It was indicated that vermicompost supplied nutrients

for peanut seedlings and plant root exudates stimulated microbial thrive, and microbial activities

stimulated nutrients release from iron mine tailing and vermicompost.

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Author Index

Abdelkrim Jawad, 165

Ablain Frédérique, 147

Aguiar Paula, 177

Aguirre Mancilla César L.,

303

Aimi Yuki, 31

Aira Manuel, 170, 171, 179,

183, 193, 271, 277, 278, 279,

280, 281, 282, 289

Ajay Neha, 300

Alarcón Gutiérrez Enrique, 54,

95, 132, 138, 141, 215, 284

Alcántar G.G., 298

Alfaro Mónica, 19

Allwood Julia, 23

Almodóvar Ana, 8, 11, 29, 34,

73

Alvarado Barcenas Estefana,

303

Alvarez Bernal Dioselina, 239,

286

Alves de Sousa Ana Paula,

244

Alves Dionicio Jair, 172

Andre Jaqne, 52, 237

Angeles Antonio, 25, 107, 176

Arancon Norman, 167, 266

Aranda Delgado Eduardo, 131,

283, 284

Arcos-Solano José M., 190

Arlei Marcela, 172

Asakawa Neuza, 135

Atuah Laura, 245

Auclerc Apolline, 91

Ávila Chi Marco Antonio, 216

Bagyaraj D.J., 212, 213, 276

Baidai Yannick, 152

Baker Geoff, 131

Balamurugan M., 48, 55, 56

Balasubramanyam Priyanka,

259

Barois Isabelle, 54, 95, 132,

138, 141, 173, 177, 190, 200,

283, 284

Barot Sebastien, 166

Barrios Edmundo, 136

Bartlam Scott, 23

Bartz Marie Luise Carolina,

96, 97, 98, 206

Bautista Francisco, 88, 216

Bayne Erin M., 168

Bearoff Frank M., 45, 66

Bellido Alain, 21, 101, 102,

113

Bello Julio, 200

Benard Y., 209

Bennett A., 52

Berdugo M., 151

Bernard Laetitia, 126

Bertrand Michel, 208

Bhattacharjee Gautam, 220

Bhattacharjee Subhalaxmi, 220

Bigorgne Emilie, 246, 252

Bilej Martin., 28, 44, 78

Binet Françoise, 162, 180, 181

Bispo A., 85

Blake Erin L., 45

Blakemore Robert, 16, 17, 18

Blanc Lilian, 143

Blanchart Eric, 35, 126

Blaxter Mark, 10, 58

Bloem Japp, 164

Blouin Manuel, 131, 210

Bolaños Martha, 135

Boothby Carlo Eduardo, 19

Boots Bas, 127

Borderas Tordesillas

Fernando, 221

Borges Sonia, 19

Botoluzzi Jeankleber, 98

Bottinelli Nicolas, 209, 217

Boubrit Sarah, 147

Boulonne L, 101

Boyer Stephane, 14, 16, 20,

129, 133, 165

Brabham P., 256

Brandstaetter Anita, 32

Brauman A., 126

Briand Marie-Paule, 181

Briard Charlène, 21, 22, 41,

102, 103

Briones Maria J.I., 36, 218,

219, 225

Brito Vega Hortencia, 173

Brown George, 57, 93, 96, 97,

98, 108, 111, 119, 192, 195,

206, 223, 247, 250, 298

Brüls T., 126

Bruford Michael W., 94, 240

Brulle Franck, 58

Brunet Didier, 211, 283

Brussaard Lijbert, 125, 130,

131, 134 Buckley Thomas R.

, 23

Bullinger-Weber G., 110, 148

Bundy Jacob G., 240

Bundy Jake, 94, 242

Burke Katherine L., 199

Butt Kevin R., 99, 112, 131,

139, 144, 205, 218, 219, 224

Callaham Mac A., 99

Cameron Erin K., 168

Campos Elizabeth, 24, 174

Cannavacciuolo Mario, 101,

102, 146

Canti Matt, 67, 128

Capowiez Yvan, 91

Caraveo Dominguez José-

Manuel, 286

Carvajal Andrés F., 105, 111

Casallas Adriana, 24

Castaneda Edana, 93

Cerdán Maria Angélica, 284

Cervantes Gabriela, 25

Chagüeza Yamileth, 135

Chandana P.G., 287

Chang Chih-Han, 15, 26, 100

Chang Yung-Ming, 70, 72

Chang-Hai Tsai, 248

Chapuis-Lardy L., 126

Charnock J.M., 52

Chaudhuri Priyasankar S., 214,

220

Chaurand, 246

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Chen Cheng-Yi, 59

Chen Jiun-Hong, 26, 51, 59,

77, 82

Chen Po-Chen, 51, 60

Chen Xu Fei, 264

Chen Yueh-Sheng, 70, 72

Chia-Wen Tsai, 248

Chih Wen-Jay, 26

Chi-Yang Huang, 248

Chotte J.L., 126

Chrozáková Alicia, 189

Chuang Shu-Chun, 51

Chung-Hsing Wang, 248

Cid-Santiago Carlos A., 226

Clause Julia, 166, 175

Cleven Denis, 9

Cluzeau Daniel, 21, 22, 41, 85,

101, 102, 103, 113, 131, 146,

147, 197, 209, 217, 273

Cocquerelle Claude, 58

Contreras Ramos Silvia

Maribel, 239, 297

Cooper Edwin L., 43, 48, 53,

55, 56, 61, 64, 70, 72, 81, 248

Cordón Pina Andrea, 230

Córdova Avalos Victor, 227

Cortesía Adriana, 255, 299

Covarrubias Prieto Jorge, 303

Coviella Carlos, 104

Cristhy Buch Andressa, 57,

247

Crittenden P.S.J., 134

Cruickshank Robert H., 165

Csuzdi Csaba, 35, 87, 100, 150

Cué Karina, 176

Cunha Luis, 177

Da Silva Elodie, 108

Dai Guohua, 268

Dai Jun, 131, 264

Da-Tian Bau, 248

Davidson Seana K., 159, 178

Davies M.S., 256

De Almeida Schiavon Greice,

301

De Andréa Mara Mercedes,

244

De Freitas Melo Vander, 250

De la Cruz Elizondo

Yadeneyro, 285

De León González Fernando,

221

De lima Ana Claudia

Rodrigues, 201, 202

De los Santos Bailon Martin,

95, 132

De Oliveira Tatiana, 208

Decaëns Thibaud, 12, 111,

122, 166, 169, 175

Degen C., 235

Delhaye Thomas, 180

Dendooven Luc, 131, 229, 239

Desjardins Thierry, 93, 111

Díaz Castelazo Cecilia, 88

Díaz Cosín Dario J., 8, 11, 29,

34, 62, 63, 73

Dilmar Baretta, 192

Dinesh M.S., 249, 287

Doan Thu Thuy, 222

Domínguez Jorge, 27, 171,

179, 182, 183, 193, 271, 277,

278, 279, 280, 281, 282, 289,

296

Donnelly Robert, 94, 238, 240

Donnison Iain, 149

Doohan Fiona M., 160

Duarte Ana Paula, 223, 250

Dulla Glen, 178

Durocher Simon, 155

Dvorak Jiri, 28, 44, 78

Edwards Clive A., 167, 266

Eisenhauer Nico, 166

El Housni H., 217

Elhottová Dana, 78, 189

Elsworth Ben, 9, 10, 58

Engelmann Péter, 61

Ernani Schwengber José, 301

Erséus Christer, 12

Espinoza Victoria David, 173

Esteller Alberich Maria

Vicenta, 275

Estrada-Segundo Margarita,

190

Exbrayat Jean-Marie, 246,

252, 253

Faber Jack, 92

Falco Liliana, 86, 104

Fang Weilin, 49, 203

Farrar Kerrie, 149

Feijoo Alexander, 105, 111

Fernández Marchán Daniel, 63

Fernández Rosa, 8, 11, 29, 34,

62, 63, 73, 186

Filley Timothy, 100

Fisher P., 52

Flores Lorena, 292

Flores Macías Medina

Antonio, 221

Flores Magallon Rebeca, 286

Fonte Steven, 136

Forey Estelle, 166, 175

Foucaud, 246

Fournier Bertrand, 106

Fragoso Carlos, 24, 25, 107,

174, 176, 221

France André-Jean, 180, 181

Francis Joe W., 43, 64, 81

Fraser Anne, 234

Freitas Barrigossi José

Alexandre, 206

Frouz Jan, 153

Fuller-Espie Sheryl L., 45, 65,

66

Fuu-Jen Tsai, 248

Gallardo Valdez Juan, 297

Gallego P.P., 225

Galsomies L., 85

Gamou Shinobu, 30, 31

Gangwar Geeta R., 300

García Hernández Gabriel, 285

Garcia Pérez José Antonio, 54,

95, 132

García Robles Marisol, 88

Garczyska Mariola, 295

Gassen Dirceu N., 206

Gbakpa Edgard A., 152

Geetha K.S., 249, 287

Geissen Violette, 229

Germain Philippe, 273

Giannopoulos Georgios, 137

Gicquel Aurélien, 180, 181

Giribet Gonzalo, 11

308


5th to 10


Giusti-Miller Stéphannie, 200

Go Ruth, 159

Gobat J.M., 110, 148, 235

Gobi Muthukaruppan, 251,

288

Godoi de Lima Odair, 108

Godoy Roberto, 229

Gómez Brandón Maria, 182,

183, 277, 278, 280, 289, 296

Gómez Vásquez Armando,

173

Gomez-Tagle Alberto (Jr.),

138

Gond Valery, 93

Gonzalez C. Korina, 284

González Castellanos

Anacleto, 290

González Dolores, 24

González Nicanor Araceli, 290

González Porto Pablo, 171,

179, 296

Govers Gerard, 124, 154, 230

Grabara Karolina, 9

Grabczysnka Olga, 184, 185

Grand C., 85

Grdisab M., 55

Grenon Cascales Graciela

Noemi, 290

Grigoropoulou Niki, 139

Grimaldi Michel, 93, 111

Gruau Gérard, 180

Guéi Arnauth M., 87, 152

Guenat C., 110, 148

Guernion Muriel, 21, 22, 41,

85, 101, 102, 103, 113

Guérold François, 91

Guerrero-Peña Armando, 262

Guevara Edward, 93

Guiller Annie, 21

Gunasekaran Paramasamy,

251, 288

Gunning P., 257

Guo Feifan, 269

Gutiérrez Mónica, 8, 11, 151,

186

Gutiérrez Rodríguez

Francisco, 221

Guzmán Jacob Valeria, 285

Gwilliam Kerry, 67, 128

Haimi Jari, 187

Hallaire Vincent, 147, 180,

197, 209, 217

Halmepuro Anne-Mari, 6

Han Dong-yun, 231, 305

Harper Georgina, 94

Harrison Kent, 167

Hättenschwiler Stephan, 143

Haubruge Eric, 163

Hebert Paul D.N., 12

Hedde M., 85

Heinen M., 134

Henry-des-Tureaux Thierry,

211

Hermy Martin, 124, 154, 230

Hernández Gustavo, 177

Hernández Hernández

Christian Arturo, 54

Hernández Juany, 255, 299

Herrera Arreola Guillermo,

286

Hlava Jakub, 89

Hlavova Andrea, 291

Hodson Mark E, 67, 128, 131,

234, 236, 245, 261

Holyoake Andrew, 165

Homa Joanna, 68, 69, 75

Howitt Robyn, 23

Huang Chih-Yang, 70, 72

Huang Kai, 231

Huerta Esperanza, 140, 216,

229, 292

Huguier Pierre, 91

Hurme Timo, 194

Hurtado Ma. Del Pilar, 111

Hutchings Tony, 236

Hynst Jaroslav, 153

Ilieva-Makulec Krassimira,

184, 185

Imery Buiza José, 293

Ingram K., 256

Islas Espinoza Marina, 275

Ivask Mari, 109

Jacob Marc, 76

Jaensch Stephan, 115

Jahnke Dênnis Silveira, 201,

202

James Samuel, 5, 12, 15, 23,

26, 159

Jarquin Aaron, 140, 229, 292

Javier Sigrid, 140, 292

Jayakumar M., 294

Jesús Juan B., 62, 73

Jiang Jibao, 41

Jiang Zhonghe, 142

Jianyang Liu, 167

Jolivet C., 102

Joner E., 246, 252, 253

Joskova R., 28, 44, 78

Josseli Nathalie, 181

Jouquet Pascal, 211

Juen Anita, 32

Juma Gabriela, 94

Jurandy Bran Nogueira

Cardoso Elke, 192

Kale Radha D., 50, 213, 249,

259, 272, 276, 287

Kam-Rigne Laossi, 166

Karmegam Natchimuthu, 294,

302

Kati Saarinen, 187

Kaur Arvinder, 267, 270

Kauschke E., 44

Kavitha B., 212

Keith Aidan M., 127

Kernecker Maria L., 155, 188

Ketoja Elise, 187

Kille Peter, 9, 10, 52, 94, 243,

257

Kintz Laura, 199

Klarica Jasmin, 32

Klimek Beata Anna, 74, 258

Klimek Malgorzata, 69, 74,

75, 258

Kohler-Milleret R., 235

Kostecka Joanna, 224, 295

Koubová Anna, 78, 189

Kramer Koen, 92

Kruk Jerzy, 69, 74, 75, 258

Kubo-Murata Makiko, 31

Kuo Wei-Wen, 70

Kuriaska Ligia, 258

309


5th to 10


Kuu Annely, 109

Labille Jérôme, 246, 252

Lago M.C.F., 225

Lambkin Denise, 67, 128, 234

Lamotte Maxime, 33

Landa Guerrero Yeudiel, 95,

132, 141

Landa Ivan, 291

Landrain Brigitte, 273

Lange Marieke de, 92

Langlois Estelle, 166, 175

Lapied Emmanuel, 143, 246,

252, 253

Lara Liliana, 190

Lara-González Rogelio, 228

Lavelle Patrick, 3, 33, 93, 111,

131, 135, 173, 200, 264, 283

Layton Caroline, 67, 128

Lazcano Cristina, 279, 281,

282, 296

Le Bayon Claire, 106, 110,

148, 235

Le Guillou C., 209

Lecomte Marcel, 273

Lee Martin R., 234

Legname E., 126

Legras Marc, 169

Li Bin-Yu, 71

Li Chang, 47

Li Fu-ping, 231, 305

Li Huan-Huan, 191, 264

Li Jing, 268

Li Wenlii, 142

Li Yang, 197, 241, 254

Liao Jyun-Ming, 26

Liao Yan, 207

Lima Ana Claudia R., 206

Liu C., 197

Liu Jer-Yuh, 72

Liu Xuelian, 79, 80, 161

Lopez C. Magdalena, 284

López López Alberto, 297

López Valeria, 107

López-Hernández Dante J.,

226

Lores Aguín Marta, 182, 183,

289

Lowe Christopher N., 76, 99

Lu Zhenyu, 142

Lubbers Ingrid, 125, 130, 164

Lunardi Marilene de Fátima,

172

Mackay A.D., 123

Maddi Priyadarshini P., 272

Magaña Mauricio, 140, 292

Makulec Grzegorz, 184

Mancikova V., 78

Marcano Elena, 255, 299

Marcano Leida, 255, 299

Margerie Pierre, 166, 175

Marichal Raphael, 93, 111

Mariño Fuencisla, 27

Martínez Rosa, 299

Martínez T. Francisco, 284

Martin-Laurent Fabrice, 162

Martins Azevedo Trigo

Priscila, 192

Matera V., 235

Mathieu Jérôme, 169

Mazur Agnieszka Irena, 46, 69

McCormick Melissa, 100

Meléndez Ramírez Virginia,

216

Mello Ivo, 98

Menasseri-Aubry S., 209, 217

Meusnier Isabelle, 12

Miambi Edouard, 200

Mikola Juha, 194

Mikulova Vlastimila, 291

Minor M.A., 123

Minutillo Madeleine M., 45

Mitchell Edward A.D., 106

Mo Xiaohui, 241, 254

Momo Fernando, 86, 104

Monard Cécile, 162

Monroy Fernando, 193, 271

Montazam Anna, 9

Montiel Rafael, 177

Mora Philippe, 200

Morales Chávez Rafael, 138

Morgan John, 9, 10, 46, 52,

58, 68, 69, 74, 75, 94, 177,

237, 240, 243, 256, 257

Mosselmans Fred, 234, 257

Moudilou Elara, 252, 253

Moussay Mickaël, 113

Muys Bart, 89, 124, 154, 230

Mysłajek Robert W., 90

Nacarelli Thimoty, 45

Nahmani Johanne, 91, 143,

246, 252

Nair Preetha, 50

Nath Sabyasachi, 214

Nava Rodríguez Verónica, 221

Nebert D. Lucas, 164

Nederström-Alanko Aime, 187

Németh Péter, 61

Nieminen Mervi, 194

Nikliska Maria, 258

Noa-Carrazana Juan C., 226

Novo Marta, 8, 11, 29, 34, 62,

63, 73, 151

Nuutinen Visa, 112, 144, 187,

194

O‘Reilly M., 52, 256

Omodeo Pietro, 33

Opper Balázs, 61

Orre Sofia, 129

Ortiz Ceballos Angel I., 95,

145, 190, 195, 196, 226, 227,

228, 262

Ortiz-Ceballos Gustavo C.,

226

Oswald Johan, 93, 111

Oughton Deborah H., 252, 253

Pablo A.L., 126

Palacios Vargas José G., 196

Palma Arriola O.B., 132

Palma-López David J., 227

Palumbo-Roe Barbara, 261

Paoletti Maurizio G., 84

Parthasarathi K., 48, 55, 56

Pasantes J.J., 36

Pascacio Velázquez José M.,

145, 196

Pasini Amarildo, 96, 97, 98,

206

Patrón I. José Cinco, 298

Pattnaik Swati, 260

Paul B., 125

Pauletti Volnei, 223, 250

310


5th to 10


Payán Zelaya Fidel, 221

Pedretti Júnior Carlos, 206

Pereira Pilecco Osivan, 98

Pérès Guénola, 21, 41, 85,

101, 102, 103, 113, 131, 146,

147, 197

Perez Hernandez Amalia, 290

Pérez Losada Marcos, 27

Pérez-García C., 36

Perroni Ventura Yareni, 54

Piacentini de Andrade Daniel,

108

Piearce Trevor, 67, 128

Pineda P. J., 298

Pinto Juma Gabriela A., 240

Piotrowska Elzbieta, 74

Piron Denis, 85, 101, 102, 113,

146, 147, 197

Pitz Scott, 100

Pizl Vaclav, 28, 78, 114, 117,

189

Plytycz Barbara, 46, 68, 69,

74, 75, 237, 258

Podolak Agnieszka, 74

Polo Aldo, 255, 299

Ponce Alejandro, 140, 292

Porco David, 12

Potthoff Martin, 13

Powell Ryan, 159

Prabha M.S. Swayam, 259

Prada Diana, 23

Prakash Om, 37

Praxedes Catarina, 93, 111

Priebe Holz Fabiana, 301

Prieto Roberto, 155, 198

Prochazkova P., 28, 44, 78

Puga-Freitas Ruben, 210

Pulleman Mirjam, 134, 137

Qiao Yuhui, 203, 241, 254

Qiu Jiang-Ping, 21, 22, 41, 103

Quintero Heimar, 105

Quintero L. R., 298

Ramírez Gómez Salvador, 227

Ramos Espinosa Guadalupe,

221

Ranganathan L.S., 48, 55, 56

Ransom Tami, 199

Rawgol Yatheesh K., 272

Raya Perez Juan Carlos, 303

Razafimbelo T., 126

Razafindrakoto Malalatiana,

35

Rearick Daniel, 199

Reddy Shobha A., 213

Reddy Vikram M., 260

Regina Cristiina, 194

Regnier Emilie, 167

Reinecke AJ, 263

Reinecke SA, 263

Richard Benoit, 12, 169

Ricoy Maigualida, 27

Rivera Zamora Raymundo,

303

Robin Paul, 273

Rodius, 246

Rodrigues Armindo, 177

Rodríguez Dioni Abril, 285

Roembke Joerg, 115, 233

Roger-Estrade Jean, 208

Romero López Angel Alonso,

215

Rosas Miguel Angel, 221

Rosciszewska Elzbieta, 69

Rose, 246

Rossmann P., 44

Roualt J., 197

Rougé Laurence, 21, 22, 85,

101, 102, 103

Rougerie Rodolphe, 12

Rowson M., 256

Roy Virginie, 200

Rozen Anna, 90, 116

Ruiz Dario, 111

Rup Pushpinder Jai, 267

Russell David, 115

Ru-Yin Tsai, 248

Saja Luth, 273

Salmon S., 186

Salomé Clémence, 110, 148

Samaritani Emanuela, 106

Sampedro Luis, 155

Sánchez Velásquez Lazaro

Rafael, 145, 228

Sánchez-Conde O., 36

Sánchez-Cortéz Mayra T., 195

Sanchez-Nuñez Minerva, 286

Sandhu Harpinder, 129

Sandler Rosana, 86, 104

Sandoval V. M., 298

Santos R. Adriana, 284

Sarrazin Max, 93, 111

Sasaki Takashi, 30

Saura Anssi, 6

Scheffczyk Adam, 115

Scheu Stefan, 166

Schiavon Greice de Almeida,

201, 202

Schiedeck Gustavo, 201, 202,

301

Schimann Heidy, 143

Schlaghamersky Jiri, 117

Schmidt Olaf, 76, 127, 160

Schofield Paul F., 234

Schon Nicole L., 123

Schwengber José Ernani, 201,

202

Scullion John, 149

Sechi Pierfrancesco, 7

Sekar Kuppuraj Raja, 302

Selvakumar Sharanya, 50

Sharma Bhargavi, 259

Sharma Venugopal, 272

Shen Huei-Ping, 26, 77

Sherman Rhonda, 274

Shimizu Nobuyoshi, 30

Shing Satyendra M., 300

Shipitalo Martin J., 112

Siagbé Goli, 152

Silerova M., 28, 44, 78

Simancas Bárbara, 63

Simoes Nelson, 177

Singh Jaswinder, 267, 270

Singh Satyendra M., 37

Sirén Taisto, 112

Six Johan, 136

Sizmur Tom, 236, 261

Skanta Frantisek, 44, 78

Sobczyk Lukasz, 90, 116

Solis Mejía Ludibeth, 275

Somon Silvia, 33

311


5th to 10


Sosa Capistrán Dulce Maria,

285

Sousa Jose Paulo, 233

Speratti Alicia, 155

Spurgeon David, 9, 10, 243

Stahl David A., 178

Stoopen Rometti Miguel, 138

Stromberger Mary, 127

Stürzenbaum Stephen R., 9,

10, 31, 68

Sun Jing, 41

Sun Qian, 38, 40, 47

Sun Zhenjun, 38, 47, 79, 80,

142, 156, 157, 161, 203, 207,

241, 254, 268, 269, 304

Suzuki Kohei, 30, 31

Svendsen Claus, 9, 10, 243

Szczesny Ewa, 68

Szlavecz Katalin, 26, 100, 150

Szymaski Wojciech, 74

Talavera José Antonio, 39

Tamiozzo Tiago, 98

Terhivuo Juhani, 6

Thakuria Dwipendra, 160

Thavamani Uma, 276

Thompson Marian, 9

Thonart Philippe, 163

Toledo Ramiro, 98

Tondoh Jerome E., 87, 152

Torres Jiménez Maria

Guadalupe, 54, 95, 141

Toyota Ayu, 153

Tran Duc Toan, 211

Traugott Michael, 32

Trefilova T., 44

Trejo Dora, 190

Trevor Piearce, 170

Trigo Dolores, 34, 62, 151,

186

Trujillo Capistrán Juan F., 262

Tsai Chang-Hai, 70, 72

Tsai Chu-Fa, 77

Tsai Fuu-Jen, 70, 72

Turner M., 52

Tylko Grzegorz, 69

Ulyett James, 149

Uribe López Sheila Ariany,

229

Uvarov Alexei, 185

Valckx Jan, 89, 124, 154, 230

Van der Walls Hans, 140, 292

Van Groenigen J.W., 125, 130,

137, 164

Vandenbulcke Franck, 58, 85

Vandenkoornhuyse Philippe,

162

Vander de Freitas Melo, 223

Vásquez-Morales Suria Gisela,

228

Velasquez Elena, 135, 283

Ventins Janis, 118

Verheyen Kris, 89

Verstraete Gorik, 89

Viga Adarsh Pal, 267

Vijayalakshmi G.S., 288

Villegas García José

Guadalupe, 303

Villenave C., 101

Volkmer Louise, 115

Voua Otomo P., 263

Wagner Maschio, 119

Wang Chong, 49, 79, 80, 191,

207

Wang Xing, 47

Wathelet Jean-Paul, 163

Weiner January, 116

Whalen Joann, 155, 188, 198

Wingate Jeremy, 236

Winters C., 52

Wood Todd C., 64, 81

Wratten Stephen D., 14, 20,

129, 133, 165

Wu Ya-Fan, 51, 82

Xiong Hui, 40, 304

Xu Yongli, 156, 157, 231, 305

Yao Lan, 40, 161

Yi Lou, 191

Yu Yu, 161

Yupeng Wu, 304

Zarate Reyna Paula, 138

Zavala Cruz Joel, 262

Zeng Zhengqing, 142

Zerbino Maria Stella, 120

Zhang Chi, 264

Zhang Donghui, 40

Zhang Junying, 156, 157, 231,

305

Zhang Ning, 207

Zhang Qin, 269

Zhang Yufeng, 38, 40

Zhao Qi, 41

Zheng Dongmei, 79, 80

Zicsi Andras, 150

Zirbes Lara, 163

Zolkiewicz Aleksandra, 68

Zúñiga Maria C., 105

international symposium on earthworm ecology 5 - archive

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